Yersinia spp. polypeptides and methods of use

Abstract

The present invention provides isolated polypeptides isolatable from a Yersinia spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

Parent Case Text

CONTINUING APPLICATION DATA

This application is a divisional patent application of U.S. patent application Ser. No. 11/336,706, filed on Jan. 20, 2006, which claims the benefit of U.S. Provisional Application No. 60/646,106, filed Jan. 21, 2005, each of which is incorporated herein by reference.

Description

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text filed entitled "293-00410103_SequenceListing_ST25.txt" having a size of 195 kilobytes and created on Oct. 7, 2013. Due to the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR .sctn.1.821(c) and the CRF required by .sctn.1.821(e). The information contained in the Sequence Listing is incorporated by reference herein.

BACKGROUND

There are three Yersinia species that are pathogenic to humans: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis is the causative agent of plague, while Y. pseudotuberculosis and specific pathogenic serovars of Y. enterocolitica cause gastrointestinal illnesses. Other species of Yersinia, including Y. rohdei, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, and Y. moolaretti, are considered enterocolitica-like opportunist pathogens with the ability to cause diarrheal illness in susceptible individuals (Agbonlahor, J Clin Microbiol, 23, 891-6, (1986), Cafferkey, et al., J Hosp Infect, 24, 109-15, (1993), Loftus, et al., Dig Dis Sci, 47, 2805-10, (2002)). The Yersinia can also infect other animal species causing a range of illnesses. Most wild and domestic species of mammals are prone to infections with the enteropathogens Y. enterocolitica and Y. pseudotuberculosis, although most of these infections are subclinical and such animals usually serve only as asymptomatic carriers of the pathogens for transmission to humans (Fantasia, et al., J Clin Microbiol, 22, 314-5, (1985), Fantasia, et al., Vet Rec, 132, 532-4, (1993), Fukushima, et al., J Clin Microbiol, 18, 981-2, (1983), Kageyama, et al., J Med Primatol, 31, 129-35, (2002), Kato, et al., Appl Environ Microbiol, 49, 198-200, (1985), Poelma, et al., Acta Zool Pathol Antverp, 3-9, (1977), Shayegani, et al., Appl Environ Microbiol, 52, 420-4, (1986), Yanagawa, et al., Microbiol Immunol, 22, 643-6, (1978).). However, there are reports that the enteropathogenic Yersinia have been associated with diarrheal illness and general malaise in domestic animals such as sheep, cattle, goats, pigs, dogs, birds and farmed deer (Jerrett, I. V., et al., Aust Vet J, 67, 212-4, (1990), Slee, K. J., et al., Aust Vet J, 65, 271-5, (1988), Slee, K. J. and C. Button, Aust Vet J, 67, 396-8, (1990), Slee, K. J. and C. Button, Aust Vet J, 67, 320-2, (1990), Zheng, X. B., J Appl Bacteriol, 62, 521-5, (1987)). Y. pestis can cause disease in a variety of rodent species as well as nonhuman primates (Davis, K. J., et al., Arch Pathol Lab Med, 120, 156-63, (1996), Meyer, K. F., et al., J Infect Dis, 129, Suppl:S85-12, (1974). Y. pestis is also associated with potentially severe infections in domestic cats (Gasper, P. W., et al., J Med Entomol, 30, 20-6, (1993)) and a few cases of Y. pestis infection have been reported in dogs (Orloski, K. A. and M. Eidson, J Am Vet Med Assoc, 207, 316-8, (1995)). In addition, Yersinia ruckeri is a pathogen of fish, causing redmouth disease in salmonids (Furones, M. D., et al., Ann. Rev. Fish Dis., 3, 105-125, (1993)).

Plague is undoubtedly one of the most devastating acute infectious disease in the recorded history of man, estimated to have killed 100 to 200 million people worldwide (Perry, R. D. and J. D. Fetherston, Clin Microbiol Rev, 10, 35-66, (1997)). In recent years plague outbreaks have been relatively uncommon in the U.S. and other industrialized countries, although endemic foci exist in all continents except Australia. Worldwide surveys indicated 2000 to 5000 annual cases of plague reported in the last several years, although epidemiologists suspect that many human cases of plague are unreported. Y. pseudotuberculosis outbreaks are fairly rare, and have occurred primarily in Finland, Japan, and the former Soviet Union (Inoue, M., et al., Zentralbl Bakteriol Mikrobiol Hyg [B], 186, 504-511, (1988), Nuorti, J. P., et al., J Infect Dis, 189, 766-774, (2004), Rodina, L. V., et al., Zh Mikrobiol Epidemiol Immunobiol, 116-118, (1998), Toyokawa, Y., et al., Kansenshogaku Zasshi, 67, 36-44, (1993)). Most Y. pseudotuberculosis infections are assumed to be transmitted by the oral-fecal route; however, a vehicle of transmission has not been identified in many cases. In the United States, infections by Y. enterocolitica are more common than those with Y. pseudotuberculosis, and are typically associated with the consumption of contaminated pork products (Ray, S. M., et al., Clin Infect Dis, 38 Suppl 3, S181-189, (2004)). The incidence of human disease caused by the Y. enterocolitica in the U.S. is difficult to determine, simply because infections associated with this organism are typically self-limiting and insufficient detection techniques have limited the ability to correctly diagnose the causative agent. However, FoodNet surveillance for 1996-1999 estimated approximately 1 case of Y. enterocolitica infection per 100,000 in the United States (Ray, S. M., et al., Clin Infect Dis, 38 Suppl 3, S181-189, (2004)).

Plague is an infectious disease of animals and humans having both enzootic and epizootic components of transmission. The most naturally occurring means of transmission is from an infected rodent reservoir to fleas, which serve as natural vectors for transmission to humans. However, human-to-human transmission can also occur by direct contact or respiratory inhalation of contaminated droplets (Pneumonic form). Nevertheless, in natural infections Y. pestis typically enter humans by a subcutaneous route into the bloodstream, where they travel to the lymph nodes and begin to multiply. Clinical manifestations of plague include large swollen masses near the lymph nodes, referred to as bubos. Occasionally, Y. pestis multiplies rapidly in the bloodstream, inducing septicemia with an accompanying general malaise that includes fever, headache, chills, and occasionally gastrointestinal disturbances. These symptoms are often misdiagnosed early, and antibiotic therapy may therefore be administered too late for effective intervention. Septicemic infection by Y. pestis has a 50% fatality rate (Perry, R. D. and J. D. Fetherston, Clin Microbiol Rev, 10, 35-66, (1997)), and can lead to pulmonary infection. The pneumonic form of plague is extremely infectious by the aerosol route and is characterized by a rapid onset of disease and a mortality rate close to 100%. Therefore, although antibiotic therapies are available and effective if administered early, the rapid onset of pneumonic plague and the misdiagnosis of septicemic plague are major obstacles in treatment of the disease.

Y. enterocolitica and Y. pseudotuberculosis are considered enteropathogens since most human infections are transmitted by the fecal-oral route and are limited to the gastrointestinal tract. In a normal host, Y. enterocolitica causes a diarrheal illness, which may be accompanied by fever and lower quadrant pain that mimics appendicitis. Y. pseudotuberculosis typically does not cause diarrheal illness, and is more likely to cause mesenteric lymphadenitis which can be misdiagnosed as appendicitis. Following ingestion, both organisms attach to the intestinal lymphoid tissues and traverse the mucosal layer, where they can subsequently multiply in the mesenteric lymph nodes and migrate to the spleen and liver (Lian, C. J., et al., J Med Microbiol, 24, 219-226, (1987), Une, T., Microbiol Immunol, 21, 505-516, (1977)). Y. pseudotuberculosis and some serotypes of Y. enterocolitica can also spread to the vascular system and cause fatal cases of septicemia (Bottone, E. J., Clin. Microbiol. Rev., 10, 257-276, (1997), Lenz, T., et al., J Infect Dis, 150, 963, (1984)), although these more invasive infections are typically limited to susceptible individuals. Y. enterocolitica has also been associated with septicemia following blood transfusions; in these cases, the blood supply was contaminated with the organism, which can survive and grow at refrigeration temperatures (Natkin, J. B., K G, Clin Lab Med, 19, 523-536, (1999)). Furthermore, intestinal Yersinia infections can lead to delayed sequelae such as reactive arthritis and thyroiditis (Bottone, E. J., Clin. Microbiol. Rev., 10, 257-276, (1997), Gaston, J. S., et al., Arthritis Rheum., 42, 2239-2242, (1999), Taccetti, G., et al., Clin Exp Rheumatol, 12, 681-684, (1994)). Antibiotic therapy has not been demonstrated to reduce the severity or duration of gastrointestinal illness caused by these two pathogens (Hoogkamp-Korstanje, J., J Antimicrob Chemother, 20, 123, (1987), Pai, C. H., et al., J Pediatr, 104, 308-11, (1984)). However, a susceptible host is typically treated with antibiotics to prevent more serious clinical manifestations of disease. Septicemia caused by either of these enteropathogens is also generally treated with antibiotics, and such therapies are frequently successful against Y. enterocolitica (Gayraud, M., et al., Clin Infect Dis, 17, 405-10, (1993)). In contrast, antibiotic therapy has traditionally been less effective in patients where septicemia is caused by Y. pseudotuberculosis, and the mortality rate associated with Y. pseudotuberculosis septicemia is approximately 75% (Natkin, J. B., K G, Clin Lab Med, 19, 523-536, (1999)).

Although natural infection by Y. pestis is rare in this country, there is fear that the organism will become a bioterrorism agent. As a tool of deliberate mass infection, the Y. pestis organism is a prime candidate due to several characteristics. First, the organism is highly infectious when spread by aerosol, a convenient method of mass dissemination. Second, there is a high mortality rate associated with Y. pestis infection if left untreated, and the pneumonic form of plague is distinguished by a rapid onset of symptoms that may be recognized too late for an effective intervention. Finally, Y. pestis has a well-defined genetic system, thus antibiotic-resistant strains are relatively easy to engineer.

Several plague vaccines with varying levels of efficacy and safety have been investigated. One of the earliest vaccines consisted of killed whole cells (KWC) of Y. pestis; this type of vaccine was first used in the late 1890's and confers protection against the bubonic form of plague. However, there is evidence that KWC immunizations offer little protection against pneumonic plague (Cohen, R. J. and J. L. Stockard, JAMA, 202, 365-366, (1967), Meyer, K. F., Bull World Health Organ, 42, 653-666, (1970)), and an additional drawback to these vaccines is that multiple injections over several months are required for protective immunity. An attenuated strain of Y. pestis, strain EV76, has been studied as a live vaccine for plague. In mouse studies, this vaccine has been shown to protect against both subcutaneous and inhalation challenges and requires as few as one dose for protection (Russell, P., et al., Vaccine, 13, 1551-1556, (1995)). However, strain EV76 is not fully avirulent, causing death in approximately 1% of vaccinated mice (Russell, P., et al., Vaccine, 13, 1551-1556, (1995)). Interestingly, there have been several unsuccessful attempts to create an avirulent strain of Y. pestis suitable for use as a live vaccine (Titball, R. W. and E. D. Williamson, Vaccine, 19, 4175-4184, (2001)).

Subunit vaccines are considered to be the most promising type of vaccine for safe and effective prevention of plague, primarily because there is no fear of adverse effects in a human host. Several surface proteins associated with Yersinia virulence were tested for their immunogenicity; all of these proteins induced an antibody response but only the F1 capsule and the secreted V antigen elicited good protection against challenge (Titball, R. W. and E. D. Williamson, Vaccine, 19, 4175-4184, (2001)). Both F1 and V antigen provide protection as individual antigens in animal models, although the combination of the two antigens provides superior protection. Many recent studies have tested F1/V vaccines formulated with alternative adjuvants in an attempt to find the best delivery system for the F1 and V antigens (Alpar, H. O., et al., Adv. Drug Deliv. Rev., 51, 173-201, (2001), Eyles, J. E., et al., J Control Release, 63, 191-200, (2000), Jones, S. M., et al., Vaccine, 19, 358-366, (2001), Reddin, K. M., et al., Vaccine, 16, 761-767, (1998), Williamson, E. D., et al., Vaccine, 19, 566-571, (2000), Williamson, E. D., et al., Vaccine, 14, 1613-9, (1996)).

Other innovative strategies have used attenuated Salmonella strains as vaccine carriers for Y. pestis antigens. When a Salmonella aroA mutant expressing an F1/V fusion protein was used as a vaccine strain, 86% of mice survived a subsequent lethal challenge dose of Y. pestis (Leary, S. E., et al., Microb Pathog, 23, 167-179, (1997)). Similarly, a vaccine consisting of a DNA plasmid bearing a gene encoding truncated-F1 capsule provided 80 to 100% protection in different mouse strains (Grosfeld, H., et al., Infect Immun, 71, 374-383, (2003)). In addition, a group of investigators mapped the B- and T-cell epitopes of the F1 antigen and utilized the immunoreactive peptides in vaccine formulations (Sabhnani, L., et al., FEMS Immunol Med Microbiol, 38, 215-29, (2003)). Their results indicated that a mixture of epitopic peptides protected 83% of mice against a lethal dose of Y. pestis.

In contrast to the extensive search for protective plague vaccines, very little research efforts have been focused on preventing infections by the enteropathogenic Yersinia species. However, a few studies have demonstrated promising results. For example, attenuated Y. enterocolitica strains administered orally to mice displayed protective effects, reducing the bacterial load in the spleen and liver following oral challenge (Igwe, E. I., et al., Infect Immun, 67, 5500-5507, (1999)). However, these strains were engineered primarily as live oral vaccine carriers, and no further testing of these strains for prevention of yersiniosis has been reported. Two subunit vaccines were demonstrated as effective in animal models of infection. The first consisted of cellular extracts from Y. enterocolitica and was administered intranasally to mice. The immunized mice demonstrated enhanced clearance of an intranasal challenge dose of Y. enterocolitica from the lungs (Di Genaro, M. S., et al., Microbiol. Immunol., 42, 781-788, (1998)). A second subunit vaccine was formulated using a heat shock protein HSP60 from Y. enterocolitica adjuvanted with interleukin-12 (Noll, A. and AutenriethIb, Infect Immun, 64, 2955-2961, (1996)). Immunizations with this vaccine resulted in significantly fewer bacteria in mouse spleens following challenge, illustrating a protective effect. Additional work utilized a vaccine consisting of DNA encoding the Y. enterocolitica HSP60 in intramuscular immunizations in mice (Noll, A., et al., Eur J Immunol, 29, 986-996, (1999)). This study demonstrated that hsp60 mRNA was present in various host tissues following immunization, but protection against Y. enterocolitica challenge was limited to the spleen and no protection was observed in the intestinal mucosa.

The similarities and differences between the diseases caused by the pathogenic Yersinia species have been the focus of much research in the past decade. This is partly due to several observations that suggest the pathogenic Yersinia provide a useful model of pathogen evolution. First, DNA hybridization studies and recent genomic comparisons of fully sequenced Y. pestis and Y. pseudotuberculosis strains have indicated that these two pathogens are highly related (Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101, 13826-13831, (2004), Ibrahim, A., et al., FEMS Microbiol Lett, 114, 173-177, (1993)), and it has been estimated that Y. pestis evolved from Y. pseudotuberculosis as recently as 1,500 to 20,000 years ago (Achtman, M., et al., Proc. Natl. Acad. Sci. USA, 96, 14043-14048, (1999)). However, despite their close evolutionary relationship, Y. pseudotuberculosis and Y. pestis cause very different diseases in humans. Furthermore, partial sequencing and 16s RNA hybridization studies suggested that Y. enterocolitica is more distantly related to the other pathogenic species of this genus (Ibrahim, A., et al., FEMS Microbiol Lett, 114, 173-177, (1993), Moore, R. L. and R. R. Brubaker, Int J Syst Bacteriol, 25, 336-339, (1975)), although Y. enterocolitica causes gastrointestinal infections similar to those observed with Y. pseudotuberculosis. Recent research has thus been focused on the virulence genes of the three pathogenic Yersinia species in an attempt to elucidate the different mechanisms they employ to cause disease. Mouse models have been particularly instructive in studying Yersinia pathogenesis, since all three species cause similar diseases in mice when injected intravenously, and more natural infections can be effectively simulated through oral and pneumonic challenge routes in mice.

A few virulence factors are unique to Y. pestis. These include proteins encoded on the Y. pestis plasmids pPCP and pMT, plasmids that are not found in Y. enterocolitica or Y. pseudotuberculosis. The pPCP plasmid encodes the plasminogen activator, a protein involved in rapid dissemination of bacteria into mammalian host tissues following subcutaneous injection (Sodeinde, O. A., et al., Science, 258, 1004-1007, (1992)). The pMT plasmid harbors at least two genes that aid in the infection of non-human hosts. The pMT-encoded caf1 gene is required for assembly of the F1 capsule, a factor that inhibits phagocytosis in the murine host but is not required for virulence in primates (Friedlander, A. M., et al., Clin. Infect. Dis., 21 Suppl 2, S178-181, (1995)). The murine toxin is also encoded on the pMT plasmid, and is believed to promote survival in the flea although it is not a required virulence factor in murine hosts (Hinnebusch, B. J., et al., Science, 296, 733-735, (2002), Hinnebusch, J., et al., Int J Med Microbiol, 290, 483-487, (2000)). Other differences between the species are the structures of the lipopolysaccharide (LPS) molecules produced by the yersiniae. Both Y. enterocolitica and Y. pseudotuberculosis express variable O-antigen side chains, which have been theorized to enhance survival in the gastrointestinal tract (Reeves, P., Trends Microbiol., 3, 381-386, (1995)) and may inhibit complement-mediated lysis during invasive disease (Karlyshev, A. V., et al., Infect Immun, 69, 7810-7819, (2001)). In contrast, Y. pestis has a rough LPS phenotype with no O-specific side chains due to mutations in several O-antigen biosynthesis genes (Prior, J. G., et al., Microb. Pathog., 30, 48-57, (2001), Skurnik, M. P., A; Ervela, E, Mol Microbiol, 37, 316-330, (2000)).

Interestingly, genomic sequencing projects revealed that several virulence genes present in all three pathogenic Yersinia species have acquired mutations in Y. pestis that rendered them non-functional (Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101, 13826-13831, (2004), Parkhill, J., et al., Nature, 413, 523-527, (2001)). Some of these encode invasin proteins that function during intestinal invasion in the enteropathogenic Y. enterocolitica and Y. pseudotuberculosis species, a host niche not colonized by Y. pestis (Simonet, M., et al., Infect Immun, 64, 375-379, (1996)). Other genes with lost function in Y. pestis include those involved in intermediary metabolism, and these functional losses are theorized to be part of the evolution of Y. pestis into an obligate parasitic species with the inability to survive outside the host (Parkhill, J., et al., Nature, 413, 523-527, (2001)). Research on the pathogenesis of Yersinia has largely been focused on the 70 kb virulence plasmid that is found in all pathogenic species of Yersinia. The sequence of this plasmid, called pYV in Y. pseudotuberculosis and pathogenic Y. enterocolitica and pCD1 in Y. pestis, is remarkably conserved between Y. pseudotuberculosis and Y. pestis (Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101, 13826-13831, (2004)). Accordingly, the more distantly-related Y. enterocolitica species harbors a more divergent pYV plasmid, but the virulence gene sequences are highly conserved among all three species (Hu, P., et al., J Bacteriol, 180, 5192-5202, (1998), Snellings, N. J., et al., Infect Immun, 69, 4627-38, (2001)). Focus on this plasmid began when experiments determined that the pYV plasmid is absolutely required for virulence of Yersinia, although the plasmid alone cannot restore virulence to specific avirulent strains suggesting that non-pVY genes are also involved in pathogenesis (Heesemann, J., et al., Infect Immun, 46, 105-110, (1984), Heesemann, J. and R. Laufs, J Bacteriol, 155, 761-767, (1983)). A large locus on this plasmid encodes the Ysc-Yop system, a type III secretion system and its associated effector proteins. This system was the first example of a type III secretion apparatus, now identified in many animal and plant microbial pathogens (for review, see Cornelis, G. R., Nat. Rev. Mol. Cell. Biol., 3, 742-752, (2002)). The Yersinia Yop-Ysc secretion system includes "injectisome" proteins, translocator effector proteins, and Yop effector proteins. Electron microscopy and labeling studies with various type III secretory systems revealed that the injectisome proteins form a pore spanning the cytoplasmic and outer membranes of the bacteria and project a needle-like structure from the cell surface (Blocker, A., et al., Mol. Microbiol., 39, 652-663, (2001), Kimbrough, T. G. and S. I. Miller, Proc Natl Acad Sci USA, 97, 11008-11013, (2000), Kubori, T., et al., Science, 280, 602-605, (1998), Sukhan, A., et al., J Bacteriol, 183, 1159-1167, (2001)). The translocator proteins appear to interact with host macrophages and polymorphonuclear neutrophils (PMNs), forming a pore-like structure in the host cell membrane (Neyt, C. and G. R. Cornelis, Mol Microbiol, 33, 971-981, (1999)). The assembled secretion apparatus then allows the effector Yops to be translocated across the bacterial cell membranes and injected into the host cell, where they function by interfering with various immune response pathways (Bleves, S. and G. R. Cornelis, Microbes Infect., 2, 1451-1460, (2000), Cornelis, G. R., Nat. Rev. Mol. Cell. Biol., 3, 742-752, (2002)). The yadA gene is also present on the pYV plasmid, encoding the YadA adhesin with the ability to bind and adhere to eukaryotic cells (Eitel, J. and P. Dersch, Infect Immun, 70, 4880-91, (2002), Skurnik, M., et al., Infect Immun, 62, 1252-61, (1994)). This protein only appears to be functional in the enteropathogenic Yersinia, as a frameshift mutation in the Y. pestis yadA gene renders it non-functional (Hu, P., et al., J Bacteriol, 180, 5192-5202, (1998)).

The involvement of iron in Yersinia infections has long been established. For example, iron-overloaded patients such as those afflicted with .beta.-thalassemia are highly susceptible to Yersinia infections (Farmakis, D., et al., Med. Sci. Monit., 9, RA19-22, (2003)). Furthermore, virulence could be restored in specific avirulent Y. pestis mutants by the addition of heme or heme-containing compounds (Burrows, T. W. and S. Jackson, Br. J. Exp. Pathol., 37, 577-583, (1956)). These early observations with Yersinia and other bacteria led researchers to study some of the microbial mechanisms of iron uptake. In mammalian hosts, available iron is extremely limited; intracellular iron is complexed with storage proteins, and extracellular iron is bound by the host proteins transferrin and lactoferrin. These iron-restricted conditions limit the growth of microbial invaders, thus acting as a defense barrier to infection. Many pathogens have evolved the ability to scavenge iron under these iron-poor conditions, effectively "stealing" iron from transferrin or heme-containing compounds. One of the most common mechanisms utilized by bacteria is the synthesis and secretion of siderophores, small molecules with a high affinity for iron (Andrews, S. C., et al., FEMS Microbiol. Rev., 27, 215-237, (2003)). The iron-siderophore complexes are bound by outer membrane receptors on the bacterial cell surface, and through the concerted action of outer membrane, periplasmic, and ABC transporter proteins, iron is transported into the cell. Other outer membrane receptors can directly bind heme and heme-containing compounds, scavenging the iron from these molecules. The role of several Yersinia iron uptake systems has been elucidated, while many more putative systems have been identified but not characterized.

Although Yersinia can use various siderophores produced by other bacteria and fungi to obtain iron, yersiniabactin is the only Yersinia-produced siderophore that has been detected (Baumler, A., et al., Zentralbl. Bakteriol., 278, 416-424, (1993), Rabsch, W. and G. Winkelmann, Biol Met, 4, 244-250, (1991), Reissbrodt, R. and W. Rabsch, Zentralbl Bakteriol Mikrobiol Hyg [A], 268, 306-317, (1988)). The yersiniabactin system is encoded by the ybt genes present on the chromosomal high-pathogenicity island (HPI), a locus that is associated with highly pathogenic strains of Yersinia (de Almeida, A. M., et al., Microb. Pathog., 14, 9-21, (1993), Rakin, A., et al., J Bacteriol, 177, 2292-2298, (1995)). The ybt genes encode proteins involved in the synthesis and secretion of the siderophore yersiniabactin (ybtS, irp1, irp2, ybtE, ybtT), as well as the cytoplasmic (ybtP, ybtQ) and outer membrane proteins (psn/fyuA) required for uptake of the iron-yersiniabactin complexes (Carniel, E., Microbes Infect., 3, 561-569, (2001)). Mutations in genes for yersiniabactin synthesis and/or uptake resulted in reduced Yersinia virulence in mouse models of infection (Bearden, S. W., et al., Infect. Immun., 65, 1659-1668, (1997), Brem, D., et al., Microbiology, 147, 1115-1127, (2001), Rakin, A., et al., Mol Microbiol, 13, 253-263, (1994)), indicating that this system is an important virulence factor in Yersinia pathogenesis. The nucleotide sequence of the ybt genes are at least 97% identical between the three pathogenic Yersinia species (Carniel, E., Microbes Infect., 3, 561-569, (2001), Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101, 13826-13831, (2004)), and the Y. pestis and Y. pseudotuberculosis ybt systems were demonstrated to be interchangeable (Perry, R. D., et al., Microbiology, 145 (Pt 5), 1181-1190, (1999)). These analyses indicated that the functions of these homologs are likely conserved among the three species. Furthermore, the HP1 has been discovered in various pathogenic species including some strains of E. coli, Citrobacter, and Klebsiella (Bach, S., et al., FEMS Microbiol. Lett., 183, 289-294, (2000)). The Ybt proteins expressed by these organisms are quite similar; indeed, antibodies raised against several of the Yersinia Ybt proteins recognized the corresponding proteins from the other pathogens (Bach, S., et al., FEMS Microbiol. Lett., 183, 289-294, (2000), Karch, H., et al., Infect Immun, 67, 5994-6001, (1999)). These results suggest that the acquisition of the ybt system is relatively recent among these pathogens and may have contributed to the invasive phenotypes associated with many of these serotypes.

Several additional ybt-independent iron uptake systems have been detected in Yersinia species based on mutation analysis, homology to known iron acquisition proteins, or the presence of iron-responsive regulatory elements. One such regulatory element is the "Fur box," a nucleotide sequence that binds the regulatory protein Fur when it is complexed with iron. The binding of Fe-Fur to a Fur box represses transcription of downstream promoters, and when iron becomes limiting, apo-Fur dissociates from DNA and transcription is derepressed. Fur and its homologs have been found in most species of bacteria, and regulate many genes in addition to iron uptake systems in diverse organisms (Campoy, S., et al., Microbiology, 148, 1039-1048, (2002), Horsburgh, M. J., et al., Infect Immun, 69, 3744-3754, (2001), Sebastian, S., et al., J Bacteriol, 184, 3965-3974, (2002), Stojiljkovic, I., et al., J Mol Biol, 236, 531-545, (1994)). Analysis of the Y. pestis genome identified many genes with Fur boxes upstream of their respective promoters, most of which encoded proteins with homology to known iron uptake systems (Panina, E. M., et al., Nucleic Acids Res, 29, 5195-5206, (2001)). Although few of these genes have been studied for function, several appear to encode iron-siderophore receptor proteins (omrA, irgA, itrA, ihaB, fauA) and iron ABC transporters (itsTUS, itpPTS). Since Yersinia can utilize siderophores produced by other organisms, these proteins may be responsible for the "siderophore piracy" observed with Yersinia. Such methods of iron acquisition are common among bacterial pathogens.

Several studies have elucidated the functions of other putative iron uptake systems. For example, the Hmu system of Y. pestis was demonstrated to acquire iron through the uptake of heme and heme-containing compounds (Hornung, J. M., et al., Mol Microbiol, 20, 725-39, (1996)). Although the ability to use heme as an iron source seems advantageous for a pathogen, the Y. pestis hmu mutant was fully virulent in a mouse model of infection (Thompson, J. M., et al., Infect Immun, 67, 3879-92, (1999)). A second putative heme-uptake system was identified in Y. pestis on the basis of sequence homology. The has genes of Y. pestis are homologs of the hemophore-dependent heme acquisition genes of Pseudomonas and Serratia (Rossi, M. S., et al., Infect Immun, 69, 6707-6717, (2001)). In these organisms, a hemophore (HasA) is secreted that binds heme and delivers it to bacterial surface receptors (HasR) to transport heme into the cell. The Y. pestis HasA protein was determined to be Fur-regulated, secreted, and capable of binding heme. However, a mutation in these genes had no effect on virulence in the mouse, even when a double mutant was tested (Rossi, M. S., et al., Infect Immun, 69, 6707-6717, (2001)). Therefore, the roles of the putative heme uptake systems in pathogenesis remain elusive, and may indicate that heme uptake is more important during infection of non-murine hosts.

The functions of two putative iron ABC transport systems have also been studied in Yersinia. The Yfe system can transport iron and manganese in Y. pestis, and yfe mutants demonstrated reduced virulence in mouse models of infection (Bearden, S. W. and R. D. Perry, Mol. Microbiol., 32, 403-414, (1999)). The second putative iron ABC transporter proteins are encoded by the yfu genes, identified by the presence of an upstream Fur box (Gong, S., et al., Infect. Immun., 69, 2829-2837, (2001)). When expressed in E. coli, the yfu genes restored growth in iron-poor media; however, comparable studies in Y. pestis failed to determine a role for Yfu in iron acquisition, and the yfu-mutant showed no defect in mouse virulence (Gong, S., et al., Infect. Immun., 69, 2829-2837, (2001)).

SUMMARY OF THE INVENTION

The present invention provides a composition including two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 40 kDa, 38 kDa, or 37 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersinia enterocolitica when incubated in media containing an iron chelator and not isolatable when grown in the media without the iron chelator. In some aspects, the composition may include two different 83 kDa polypeptides isolatable from a Y. enterocolitica when incubated in media comprising an iron chelator. The composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729. The composition may further include an isolated polypeptide having a molecular weight of 268 kDa, 92 kDa, 79 kDa, 54 kDa, 45 kDa, 31 kDa, 28 kDa, or a combination thereof, and isolatable from a Y. enterocolitica when grown in the media without the iron chelator.

The present invention also provides a composition including two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 268 kDa, 79 kDa, or 45 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersinia enterocolitica when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. The composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729.

The present invention further provides a composition including isolated polypeptides having molecular weights of 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, and 28 kDa, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides are isolatable from a Yersinia enterocolitica, and the composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729.

The present invention provides a composition including two isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 46 kDa, 37 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa are isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. The composition protects a mouse against challenge with Y. pestis strain KIM6+. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729. The composition may further include an isolated polypeptide having a molecular weight of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa, and isolatable from a Y. pestis when grown in the media without the iron chelator. The polypeptides may be isolatable, or in some aspects isolated from Y. pestis strain KIM6+.

The present invention also provides a composition including two isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, 20 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, are isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. The composition protects a mouse against challenge with Y. pestis strain KIM6+. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. pestis strain KIM6+.

The present invention further provides a composition including isolated polypeptides having molecular weights of 254 kDa, 104 kDa, 99 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 60 kDa, 46 kDa, 44 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, and 20 kDa wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides are isolatable from a Yersinia pestis, and the composition protects a mouse against challenge with Y. pestis strain KIM6+. The polypeptides may be isolatable, or in some aspects isolated from Y. pestis strain KIM6+.

The present invention provides a method for treating in infection in a subject including administering an effective amount of a composition of the present invention to a subject having or at risk of having an infection caused by a Yersinia spp. The subject may be an animal, such as a fish or a mammal, such as a human. The Yersinia spp. may be, for example, Y. enterocolitica or Y. pestis, or Y. ruckeri.

The present invention also provides a method for treating a symptom in a subject including administering an effective amount of a composition of the present invention to a subject having an infection caused by a Yersinia spp. The subject may be an animal, such as a fish or a mammal, such as a human. The Yersinia spp. may be, for example, Y. enterocolitica or Y. pestis, or Y. ruckeri. The symptom may be, for example, diarrhea, enteritis, plague, red mouth disease, or a combination thereof.

The present invention further provides for treating in infection in a subject including administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Yersinia spp., wherein the composition includes antibody that specifically binds a polypeptide of the present invention. The antibody may be polyclonal or monoclonal. In one example, the antibody specifically binds two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, wherein the polypeptides are isolatable from a Yersinia enterocolitica when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. In another example, the antibody specifically binds two isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, wherein the polypeptides are isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator.

The present invention also provides a method for treating a symptom in a subject including administering an effective amount of a composition to a subject having an infection caused by a Yersinia spp., wherein the composition includes antibody that specifically binds a polypeptide of the present invention. The antibody may be polyclonal or monoclonal. In one example, the antibody specifically binds two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel, wherein the polypeptides are isolatable from a Yersinia enterocolitica when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. In another example, the antibody specifically binds two isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, wherein the polypeptides are isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator.

The present invention further provides kits for detecting antibody that specifically binds a polypeptide of the present invention. The kit includes an isolated polypeptide of the present invention, and a reagent that detects an antibody that specifically binds the polypeptide. The polypeptide and the reagent are typically present in separate containers. In one example, the polypeptide may have a molecular weight of 83 kDa, 70 kDa, or 66 kDa, or a combination thereof, wherein the polypeptide is isolatable from a Yersinia enterocolitica when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. In another example, the polypeptide may have a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, wherein the polypeptide is isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Detergent-insoluble protein-enriched extracts of Y. enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ resolved by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel. The numbers to the left of the gel image denote the molecular weights in kDa of the standards shown in Lane 1. Lane 1, molecular weight standards; Lane 2, Y. pestis strain KIM6+ grown in media supplemented with 300 .mu.M FeCl.sub.3; Lane 3, Y. pestis strain KIM6+ grown in media supplemented with 160 .mu.M 2,2-diprydyl; Lane 4, Y. enterocolitica ATCC strain 27729 grown in media supplemented with 160 .mu.M 2,2-diprydyl; Lane 5, Y. enterocolitica ATCC strain 27729 grown in media supplemented with 300 .mu.M FeCl.sub.3.

FIG. 2. Survival of vaccinated and non-vaccinated mice following challenge with Y. enterocolitica. Chart showing survival analysis of mice following immunization with membrane proteins derived from Y. enterocolitica strain 27729 grown under iron-limiting conditions and subsequent live challenge with strain 27729. Mortality was recorded for 7 days following challenge.

FIG. 3. Nucleotide sequences of SEQ ID NOs: 1-23.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions including polypeptides. As used herein, "polypeptide" refers to a polymer of amino acids linked by peptide bonds. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. The term polypeptide does not connote a specific length of a polymer of amino acids. A polypeptide may be isolatable directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. In the case of a polypeptide that is naturally occurring, such a polypeptide is typically isolated. An "isolated" polypeptide is one that has been removed from its natural environment. For instance, an isolated polypeptide is a polypeptide that has been removed from the cytoplasm or from the outer membrane of a cell, and many of the polypeptides, nucleic acids, and other cellular material of its natural environment are no longer present. An "isolatable" polypeptide is a polypeptide that could be isolated from a particular source. A "purified" polypeptide is one that is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Polypeptides that are produced outside the organism in which they naturally occur, e.g., through chemical or recombinant means, are considered to be isolated and purified by definition, since they were never present in a natural environment. As used herein, a "polypeptide fragment" refers to a portion of a polypeptide that results from digestion of a polypeptide with a protease. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

A polypeptide of the present invention may be characterized by molecular weight, mass fingerprint, or the combination thereof. The molecular weight of a polypeptide, typically expressed in kilodaltons (kDa), can be determined using routine methods including, for instance, gel filtration, gel electrophoresis including sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis, mass spectrometry, and liquid chromatography including HPLC. Preferably, molecular weight is determined by resolving a polypeptide using an SDS polyacrylamide gel having a stacking gel of about 4% and a resolving gel of about 10% under reducing and denaturing conditions. Unless indicated otherwise, molecular weight refers to molecular weight as determined by SDS-PAGE. As used herein, a "mass fingerprint" refers to a population of polypeptide fragments obtained from a polypeptide after digestion with a protease. Typically, the polypeptide fragments resulting from a digestion are analyzed using a mass spectrometric method. Each polypeptide fragment is characterized by a mass, or by a mass (m) to charge (z) ratio, which is referred to as an "m/z ratio" or an "m/z value". Methods for generating a mass fingerprint of a polypeptide are routine. An example of such a method is disclosed in Example 9.

Polypeptides of the present invention may be metal regulated polypeptides. As used herein, a "metal regulated polypeptide" is a polypeptide that is expressed by a microbe at a greater level when the microbe is grown in low metal conditions compared to growth of the same microbe in high metal conditions. Low metal and high metal conditions are described herein. For instance, one class of metal regulated polypeptide produced by Yersinia spp. is not expressed at detectable levels during growth of the microbe in high metal conditions but is expressed at detectable levels during growth in low metal conditions. Examples of such metal regulated polypeptides isolatable from Yersinia enterocolitica have molecular weights of 83 kDa, 70 kDa, or 66 kDa. In some aspects, Y. enterocolitica may produce two different polypeptides each having a molecular weight of 83 kDa and each expressed at detectable levels during growth of the microbe in low metal conditions and not expressed at detectable levels during growth in high metal conditions. Examples of such metal regulated polypeptides isolatable from Yersinia pestis have molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa.

Another type of metal regulated polypeptide produced by Yersinia spp. is expressed at detectable levels during growth of the microbe in high metal conditions but significantly more of the polypeptide is expressed during growth in low metal conditions. The expression of such polypeptides is referred to herein as "enhanced" during growth in low metal conditions. Typically, the expression of a polypeptide during growth in low metal conditions is at least 10% or at least 50% greater than the expression of the polypeptide during growth in high metal conditions. Examples of metal regulated polypeptides showing enhanced expression and isolatable from Y. enterocolitica have molecular weights of 268 kDa, 79 kDa, or 45 kDa. Examples of metal regulated polypeptides showing enhanced expression and isolatable from Y. pestis have molecular weights of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa. In some aspects, Y. pestis may produce two different polypeptides each having a molecular weight of 31 kDa and each showing enhanced expression.

The expression of some polypeptides of the present invention is not significantly influenced by the presence of a metal. Examples of such polypeptides isolatable from Y. enterocolitica have molecular weights of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa. In some aspects, Y. enterocolitica may produce two different polypeptides each having a molecular weight of 31 kDa and each not significantly influenced by the presence of a metal. Examples of such polypeptides isolatable from Y. pestis have molecular weights of 104 kDa, 99 kDa, 60 kDa, or 44 kDa.

Whether a polypeptide is a metal regulated polypeptide or not can be determined by methods useful for comparing the presence of polypeptides, including, for example, gel filtration, gel electrophoresis including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, mass spectrometry, and liquid chromatography including HPLC. Separate cultures of a microbe are grown under high metal conditions and under low metal conditions, polypeptides of the present invention are isolated as described herein, and the polypeptides present in each culture are resolved and compared. Typically, an equal amount of polypeptides from each culture is used. Preferably, the polypeptides are resolved using an SDS polyacrylamide gel having a stacking gel of about 4% and a resolving gel of about 10% under reducing and denaturing conditions. For instance, 30 micrograms (.mu.g) of total polypeptide from each culture may be used and loaded into wells of a gel. After running the gel and staining the polypeptides with Coomasie Brilliant Blue, the two lanes can be compared. When determining whether a polypeptide is or is not expressed at a detectable level, 30 .mu.g of total polypeptide from a culture is resolved on an SDS-PAGE gel and stained with Coomasie Brilliant Blue using methods known in the art. A polypeptide that can be visualized by eye is considered to be expressed at a detectable level, while a polypeptide that cannot be visualized by eye is considered to be not expressed at a detectable level.

Polypeptides of the present invention may have immunogenic activity. "Immunogenic activity" refers to the ability of a polypeptide to elicit an immunological response in an animal. An immunological response to a polypeptide is the development in an animal of a cellular and/or antibody-mediated immune response to the polypeptide. Usually, an immunological response includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed to an epitope or epitopes of the polypeptide. "Epitope" refers to the site on an antigen to which specific B cells and/or T cells respond so that antibody is produced. The immunogenic activity may be protective. "Protective immunogenic activity" refers to the ability of a polypeptide to elicit an immunological response in an animal that prevents or inhibits infection by Yersinia spp., for instance, Y. enterocolitica or Y. pestis. Whether a polypeptide has protective immunogenic activity can be determined by methods known in the art, for instance as described in example 4 or example 7. For example, a polypeptide of the present invention, or combination of polypeptides of the present invention, protect a rodent such as a mouse against challenge with a Yersinia spp. A polypeptide of the present invention may have seroreactive activity. "Seroactive activity" refers to the ability of a candidate polypeptide to react with antibody present in convalescent serum from an animal infected with a Yersinia spp., preferably Y. enterocolitica or Y. pestis. Polypeptides of the present invention may have immunoregulatory activity. "Immunoregulatory activity" refers to the ability of a polypeptide to act in a nonspecific manner to enhance an immune response to a particular antigen. Methods for determining whether a polypeptide has immunoregulatory activity are known in the art.

A polypeptide of the present invention has the characteristics of a polypeptide expressed by a reference microbe. The characteristics include both molecular weight and mass fingerprint. The reference microbe can be Y. enterocolitica, Y. pseudotuberculosis, Y. pestis, Y. ruckeri, Y. rohdei, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, or Y. moolaretti, preferably Y. enterocolitica, for instance, Y. enterocolitica ATCC strain 27729, or Y. pestis, for instance, Y. pestis strain KIM6+(Gong et al., Infect. Immun., 69:2829-2837 (2001)).

When the reference microbe is Y. enterocolitica, for instance, Y. enterocolitica ATCC strain 27729, a candidate polypeptide is considered to be a polypeptide of the present invention if it has a molecular weight of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, or 45 kDa, and has a mass fingerprint that is similar to the mass fingerprint of a metal regulated polypeptide expressed by the reference microbe and having a molecular weight of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, or 45 kDa, respectively. Preferably, such polypeptides are metal regulated. For instance, a candidate polypeptide is a polypeptide of the present invention if it has a molecular weight of 83 kDa and has a mass fingerprint similar to the mass fingerprint of one of the metal regulated 83 kDa polypeptides produced by the reference strain Y. enterocolitica ATCC strain 27729. A candidate polypeptide is also considered to be a polypeptide of the present invention if it has a molecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa and has a mass fingerprint that is similar to the mass fingerprint of a polypeptide expressed by the reference microbe and having a molecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa, respectively.

When the reference microbe is Y. pestis, for instance, Y. pestis strain KIM6+, a candidate polypeptide is considered to be a polypeptide of the present invention if it has a molecular weight of 254 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa, and has a mass fingerprint that is similar to the mass fingerprint of a metal regulated polypeptide expressed by the reference microbe and having a molecular weight of 254 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa, respectively. Preferably, such polypeptides are metal regulated. For instance, a candidate polypeptide is a polypeptide of the present invention if it has a molecular weight of 94 kDa and has a mass fingerprint similar to the mass fingerprint of one of the metal regulated 94 kDa polypeptides produced by the reference strain Y. pestis strain KIM6+. A candidate polypeptide is also considered to be a polypeptide of the present invention if it has a molecular weight of 104 kDa, 99 kDa, 60 kDa, or 44 kDa and has a mass fingerprint that is similar to the mass fingerprint of a polypeptide expressed by the reference microbe and having a molecular weight of 104 kDa, 99 kDa, 60 kDa, or 44 kDa, respectively.

The polypeptides expressed by a reference microbe and referred to above by molecular weight can be obtained by growth of the reference microbe under low metal conditions and the subsequent isolation of a polypeptide by the processes disclosed herein. A candidate polypeptide is isolatable from a microbe, preferably a gram negative microbe, more preferably, a member of the family Enterobacteriaceae preferably, a member of the genus Yersinia, such as Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis. A candidate polypeptide may also be produced using recombinant, enzymatic, or chemical techniques.

A candidate polypeptide may be evaluated by mass spectrometric analysis to determine whether the candidate polypeptide has a mass fingerprint similar to one of the polypeptides expressed by a reference microbe and referred to above by molecular weight. Typically, the candidate polypeptide is isolated, for instance by resolving the candidate polypeptide by gel electrophoresis and excising the portion of the gel containing the candidate polypeptide. Any gel electrophoresis method that separates polypeptides based on differing characteristics can be used, including 1 dimensional or 2 dimensional gel electrophoresis, as well as liquid chromatographic separation based on, for instance, hydrophobicity, pI, or size. The candidate polypeptide is fragmented, for instance by digestion with a protease. Preferably, the protease cleaves the peptide bond on the carboxy-terminal side of the amino acid lysine and the amino acid arginine, except when the amino acid following the lysine or the arginine is a proline. An example of such a protease is trypsin. Methods for digesting a polypeptide with trypsin are routine and known in the art. An example of such a method is disclosed in Example 9.

Methods for the mass spectrometric analysis of polypeptides are routine and known in the art and include, but are not limited to, matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS). Typically, a mixture containing the polypeptide fragments obtained from a candidate polypeptide is mixed with a matrix that functions to transform the laser energy to the sample and produce ionized, preferably monoisotopic, polypeptide fragments. Examples of matrices that can be used include, for instance, sinapinic acid or cyano-4-hydroxycinnamic acid. An example of a method for the analysis of polypeptides by MALDI-TOF MS is described in Example 9. The ionized polypeptide fragments are separated according to their m/z ratio, and detected to yield a spectrum of m/z ratio versus intensity. The spectrum includes m/z values that represent the polypeptide fragments derived from the candidate polypeptide. For any given polypeptide, the amount of each polypeptide fragment resulting from a trypsin digestion should be equimolar. However, it is known that trypsin digestion is not always 100% efficient, for instance, some sites are more efficiently cleaved. Thus, when MALDI-TOF MS is used to determine m/z values, the intensity of each m/z value is typically not identical. Generally, a spectrum has a background level of noise present across most of the x-axis (i.e., the axis having the values of the m/z ratios). This background level of noise varies depending on the running conditions and the machine used, and is easily identified by visual inspection of the spectrum. An m/z value is generally considered to represent a polypeptide fragment when the intensity is at least 2 times greater, 3 times greater, or 4 times greater than the background level of noise. The spectrum usually includes other m/z values that are artifacts resulting from, for instance, incomplete digestion, over digestion, other polypeptides that may be present in the mixture, or the protease used to digest the polypeptide including m/z values resulting from autolysis of the protease. This method of digesting a polypeptide with a protease is recognized by the art as resulting in a mass fingerprint of great specificity that can be used to accurately characterize the polypeptide and distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide is analyzed by mass spectroscopy, preferably both the candidate polypeptide and the polypeptide from the reference microbe are prepared and analyzed together, thereby decreasing any potential artifacts resulting from differences in sample handling and running conditions. Preferably, all reagents used to prepare and analyze the two polypeptides are the same. For instance, the polypeptide from the reference microbe and the candidate polypeptide are isolated under substantially the same conditions, fragmented under substantially the same conditions, and analyzed by MALDI-TOF MS on the same machine under substantially the same conditions. A mass fingerprint of a candidate polypeptide is considered to be similar to the mass fingerprint of a polypeptide from a reference microbe when at least 80%, at least 90%, at least 95%, or substantially all of the m/z values present in the spectrum of the reference microbe polypeptide and above the background level of noise are also present in the spectrum of the candidate polypeptide.

In another aspect, a polypeptide is considered to be a polypeptide of the present invention if it has a molecular weight of a reference polypeptide described in Table 1 or Table 2 and has a mass fingerprint that includes the population of polypeptide fragments of the reference polypeptide as listed in Table 1 or Table 2. For instance, a polypeptide of the present invention includes a polypeptide of 83 kDa and a mass fingerprint that includes polypeptide fragments having masses of 686.37, 975.45, 1000.53, 1015.46, 1140.65, 1169.68, 1170.64, 1197.57, 1342.55, 1356.74, 1394.67, 1452.73, 1476.72, 1520.76, 1692.77, 1715.75, 1828.79, 1960.91, 2013.02, 2018.95, 2040.97, 2163.05, 2225.03, 2416.19, and 3174.44, or a mass fingerprint that includes polypeptide fragments having masses of 1001.49, 1103.57, 1139.57, 1154.51, 1170.49, 1208.59, 1213.67, 1337.70, 1452.86, 1567.84, 1633.85, 1650.82, 1659.91, 1708.77, 1748.95, 1849.92, 1986.98, 2103.95, 2111.03, 2163.11, 2386.19, 2452.09, 2537.34, and 3422.66. The mass fingerprint of a candidate polypeptide can be determined by a mass spectrometric method, for instance by MALDI-TOF MS. The mass fingerprint of a candidate polypeptide will generally have additional polypeptide fragments and therefore additional m/z values other than those listed for a polypeptide in Table 1 or Table 2. Preferably, when the candidate polypeptide is being compared to a polypeptide in Table 1 or Table 2, the candidate polypeptide is obtained from a Y. pestis, Y. pseudotuberculosis, or Y. enterocolitica, more preferably, Y. enterocolitica or Y. pestis. A candidate polypeptide can be obtained by growth of a microbe under low metal conditions and the subsequent isolation of a polypeptide by the processes described herein.

It is well known in the art that modifications of amino acids can be accidentally introduced during sample handling, such as oxidation, and formation of carbamidomethyl derivatives. Further, these types of modifications alter the m/z value of a polypeptide fragment. For instance, if a polypeptide fragment contains a methoinine that is oxidized the m/z value will be increased by 16 relative to the same fragment that does not contain the oxidized methionine. Accordingly, those polypeptide fragments in Tables 1 and 2 having the notation "oxidation (M)" have an m/z value that is increased by 16 relative to the same fragment that does not contain the oxidized methionine. It is understood that the polypeptide fragments of Table 1 and Table 2 can be modified during sample handling.

TABLE-US-00001 TABLE 1 Characteristics of polypeptides obtained from Y. enterocolitica. mass of polypeptide approximate fragments molecular resulting weight in from predicted amino acid sequence of the polypeptide kilodaltons trypsin polypeptide fragment designation (kDa).sup.1 digest.sup.2 (SEQ ID Numbers listed in parenthesis) Lw545 268 928.45 FHQLDNR (SEQ ID No: 24) 1139.57 VNFTAGVGGYR (SEQ ID No: 25) 1311.65 NSVSIGHESLNR (SEQ ID No: 26) 1439.69 ASTSDTGVAVGFNSK (SEQ ID No: 27) 1525.73 SAETLASANVYADSK (SEQ ID No: 28) 1554.69 EAFDLSNDALDMAK + Oxidation (M) (SEQ ID No: 29) 1580.77 SAEVLGIANNYTDSK (SEQ ID No: 30) 1595.78 ALGDSAVTYGAGSTAQK (SEQ ID No: 31) 1682.78 EAFDLSNDALDMAKK + Oxidation (M) (SEQ ID No: 32) 2109.18 AAVAVGAGSIATGVNSVAIGPLSK (SEQ ID No: 33) Lw391A 83 686.37 DIGNIR (SEQ ID No: 34) 975.45 FFVSYQW (SEQ ID No: 35) 1000.53 VNGQDVTLR (SEQ ID No: 36) 1015.46 ASYFDTNAK (SEQ ID No: 37) 1140.65 DLPVSILAGTR (SEQ ID No: 38) 1169.68 QGVLTLVDGIR (SEQ ID No: 39) 1170.64 NIPGLTVTGSGR (SEQ ID No: 40) 1197.57 YYNNSALEPK (SEQ ID No: 41) 1342.55 APTMGEMYNDSK (SEQ ID No: 42) 1356.74 IDQIQSLSANLR (SEQ ID No: 43) 1394.67 TDDVDGILSFGTR (SEQ ID No: 44) 1452.73 GMTTTVVLGNAFDK (SEQ ID No: 45) 1476.72 IADTMVVTATGNER (SEQ ID No: 46) 1520.76 FGSGWLQDEITLR (SEQ ID No: 47) 1692.77 NPQTSAASSTNLMTDR (SEQ ID No: 48) 1715.75 FNDLMMAEDDLQFK (SEQ ID No: 49) 1828.79 GSSEGYADVDADKWSSR (SEQ ID No: 50) 1960.91 QEQTPSGATESFPQADIR (SEQ ID No: 51) 2013.02 QGTDTGHLNSTFLDPALVK (SEQ ID No: 52) 2018.95 QSDGFNAPNDETISNVLAK (SEQ ID No: 53) 2040.97 VYSAAATGDHSFGLGASAFGR (SEQ ID No: 54) 2163.05 LFTDSFASHLLTYGTEAYK (SEQ ID No: 55) 2225.03 VSSSGTPQAGYGVNDFYVSYK (SEQ ID No: 56) 2416.19 GAVSVTPTDWLMLFGSYAQAFR (SEQ ID No: 57) 3174.44 SSFEAPMMVTVVEADTPTSETATSATDMLR (SEQ ID No: 58) Lw391B 83 1001.49 NDASVQNVR (SEQ ID No: 59) 1103.57 IGFLGQQDAR (SEQ ID No: 60) 1139.57 VNLGYAANYR (SEQ ID No: 61) 1154.51 GYGNPSQNYR (SEQ ID No: 62) 1170.49 YGDDDQFGVR (SEQ ID No: 63) 1208.59 GHFDTGPITHK (SEQ ID No: 64) 1213.67 LLASATWLDPK (SEQ ID No: 65) 1337.70 NVPFNVIGYTSK (SEQ ID No: 66) 1452.86 LKPWTRLDLGVR (SEQ ID No: 67) 1567.84 VSLYANHIEALGPGK (SEQ ID No: 68) 1633.85 GIELNVFGEPVFGTR (SEQ ID No: 69) 1650.82 TNDTITVVGAQETFR (SEQ ID No: 70) 1659.91 VTPIYGIMVKPWEK (SEQ ID No: 71) 1708.77 NFDSGVPNSAGSLDAMK (SEQ ID No: 72) 1748.95 LYVPYVADSVAGLGGIR (SEQ ID No: 73) 1849.92 VTVDYGSASQVGGALDVGR (SEQ ID No: 74) 1986.98 AGGNDLIPTYLDGQVANGGR (SEQ ID No: 75) 2103.95 SEYDVSQNWTVYGSVGASR (SEQ ID No: 76) 2111.03 GYNLDGDDISFGGLFGVLPR (SEQ ID No: 77) 2163.11 SGSQYANEANTLKLKPWTR (SEQ ID No: 78) 2386.19 GANAFINGISPSGSGVGGMINLEPK(SEQ ID No: 79) 2452.09 NEETGQYGAPMLTNNNGDATISR (SEQ ID No: 80) 2537.34 SAPYQYNGKPVVNAGQIPGIIHSK (SEQ ID No: 81) 3422.66 YGGTLALFEITRPTGMVDPATNVYGFYGEQR (SEQ ID No: 82) Lw392 79 836.44 YDTVALR (SEQ ID No: 83) 1017.59 VLLGVDFQK (SEQ ID No: 84) 1070.48 FDDVWSFR (SEQ ID No: 85) 1085.50 SVQATVGYDF (SEQ ID No: 86) 1131.59 ADLGTWAASLK (SEQ ID No: 87) 1188.55 QWADDANTLR (SEQ ID No: 88) 1214.63 VNSQGLELEAR (SEQ ID No: 89) 1235.65 AVPATYYVPAGK (SEQ ID No: 90) 1255.66 LSVIAGYTYNR (SEQ ID No: 91) 1263.65 VPSYTLGDASVR (SEQ ID No: 92) 1360.66 RPQFTSEGHFR (SEQ ID No: 93) 1496.67 GFFDGESNHNVFK (SEQ ID No: 94) 1501.79 GAFVQLNVNNIADK (SEQ ID No: 95) 1614.75 WQQIYSYEFSHK (SEQ ID No: 96) 1652.77 GFFDGESNHNVFKR (SEQ ID No: 97) 1717.82 GFHGGDVNNTFLDGLR (SEQ ID No: 98) 1770.85 RWQQIYSYEFSHK (SEQ ID No: 99) 1819.86 AGHEADLPTSGYTATTTK (SEQ ID No: 100) 1827.01 TDQPLILTAQSVSVVTR (SEQ ID No: 101) 2004.92 DPSGGYHSAVPADGSIYGQK (SEQ ID No: 102) 2066.02 GPSSALYGQSIPGGVVMMTSK (EQ ID No: 103) 2119.91 KYVAACYSTSYCYWGAER (SEQ ID No: 104) 2299.22 YAIAPSLLWQPDENTSLLLR (SEQ ID No: 105) 2307.15 LLSDGGSYNVLQVDPWFLER (SEQ ID No: 106) 2782.23 QNASYTHSNTQLEQVYQGGWNSDR (SEQ ID No: 107) 2911.35 LTAGNNNTQVAAFDYTDAISEHWAFR (SEQ ID No: 108) 3023.42 RYEQSGVYLQDEMTLDNWHLNLSGR (SEQ ID No: 109) 3286.53 QQMDDQNVATVNQALNYTPGVFTGFSGGATR (SEQ ID No: 110) Lw393 70 713.42 VPFVPR (SEQ ID No: 111) 759.42 TVGINTR (SEQ ID No: 112) 806.41 YGALMPR (SEQ ID No: 113) 819.42 FDIGGGVR (SEQ ID No: 114) 919.48 GPQGTLYGK (SEQ ID No: 115) 1023.50 GYIEGGVSSR (SEQ ID No: 116) 1051.53 SINYELGTR (SEQ ID No: 117) 1186.57 WNQDVQELR (SEQ ID No: 118) 1199.60 TVDMVFGLYR (SEQ ID No: 119) 1394.68 YGAGSSVNGVIDTR (SEQ ID No: 120) 1436.66 LSLSDGSPDPYMR (SEQ ID No: 121) 1479.70 ATQDAYVGWNDIK (SEQ ID No: 122) 1540.80 INISVHVDNLFDR (SEQ ID No: 123) 1545.80 TFPSGSLIVNMPQR (SEQ ID No: 124) 1564.76 KLSLSDGSPDPYMR (SEQ ID No: 125) 1667.72 SEFTNDSELYHGNR (SEQ ID No: 126) 1730.85 FAPGWSWDINGNVIR (SEQ ID No: 127) 1789.81 LAPDDQPWEMGFAASR (SEQ ID No: 128) 1904.85 TYGYMNGSSAVAQVNMGR (SEQ ID No: 129) 1981.02 SAQGGIINIVTQQPDSTPR (SEQ ID No: 130) 1982.93 QGTYATLDSSLGWQATER (SEQ ID No: 131) 1995.94 DMQLYSGPVGMQTLSNAGK (SEQ ID No: 132) 2009.89 SSTQYHGSMLGNPFGDQGK (SEQ ID No: 133) 2027.02 LAVNLVGPHYFDGDNQLR (SEQ ID No: 134) 2058.99 LRLAPDDQPWEMGFAASR (SEQ ID No: 135) 2132.93 QVDDGDMINPATGSDDLGGTR(SEQ ID No: 136) 2162.17 FNLSGPIQDGLLYGSVTLLR (SEQ ID No: 137) 2274.20 VLPGLNIENSGNMLFSTISLR (SEQ ID No: 138) 2363.13 SEFTNDSELYHGNRVPFVPR (SEQ ID No: 139) 2377.30 SKFNLSGPIQDGLLYGSVTLLR (SEQ ID No: 140) 2383.07 SNDDQVLGQLSAGYMLTDDWR (SEQ ID No: 141) 2563.22 SASANNVSSTVVSAPELSDAGVTASDK (SEQ ID No: 142) 2657.23 YTTDDWVFNLISAWQQQHYSR (SEQ ID No: 143) 2833.50 IAQGYKPSGYNIVPTAGLDAKPFVAEK (SEQ ID No: 144) 2929.46 SASANNVSSTVVSAPELSDAGVTASDKLPR (SEQ ID No: 145) Lw550 66 867.49 VSGLLSHR (SEQ ID No: 146) 881.42 TSEYLNR (SEQ ID No: 147) 883.43 EWHGTVR (SEQ ID No: 148) 1020.59 YTLILVDGK (SEQ ID No: 149) 1086.57 RVDIEVNDK (SEQ ID No: 150) 1167.61 VGKEWHGTVR (SEQ ID No: 151) 1176.69 YTLILVDGKR (SEQ ID No: 152) 1207.63 LMGGVYNVLDK (SEQ ID No: 153) 1345.72 IQDSAASISVVTR (SEQ ID No: 154) 1748.72 MDQDENYGTHWTPR (SEQ ID No: 155) 1753.77 NEFDFDIGHYVQDR (SEQ ID No: 156) 1850.95 DVPGVVVTGGGSHSDISIR (SEQ ID No: 157) 2520.27 GTRPNSDGSGIEQGWLPPLAAIER (SEQ ID No: 158) 2606.16 NNYAITHHGYYDFGNSTSYVQR (SEQ ID No: 159) 2942.50 AYTDITDALKDVPGVVVTGGGSHSDISIR (SEQ ID No: 160) 3085.41 NGAATFTLTPDDKNEFDFDIGHYVQDR (SEQ ID No: 161) Lw552 45 1139.57 VNFTAGVGGYR (SEQ ID No: 162) 1208.61 SSQALAIGSGYR (SEQ ID No: 163) 1311.65 NSVSIGHESLNR (SEQ ID No: 164) 1439.69 ASTSDTGVAVGFNSK (SEQ ID No: 165) 1500.74 TTLETAEEHTNKK (SEQ ID No: 166) 1525.73 SAETLASANVYADSK (SEQ ID No: 167) 1580.77 SAEVLGIANNYTDSK (SEQ ID No: 168) 1595.78 ALGDSAVTYGAGSTAQK (SEQ ID No: 169) Lw555 37 704.42 LGFAGLK (SEQ ID No: 170) 880.43 ADAYSGGLK (SEQ ID No: 171) 970.38 DGDQSYMR (SEQ ID No: 172) 1121.57 DGNKLDLYGK (SEQ ID No: 173) 1279.54 AEDQDQGNFTR (SEQ ID No: 174) 1294.58 VDGLHYFSDDK (SEQ ID No: 175) 1334.67 INLLDENEFTK (SEQ ID No: 176) 1509.71 VDGLHYFSDDKSK (SEQ ID No: 177) 1907.96 NAGINTDDIVAVGLVYQF (SEQ ID No: 178) 2245.12 NTNFFGLVDGLNFALQYQGK (SEQ ID No: 179) 2324.11 YDANNVYLAATYAQTYNLTR (SEQ ID No: 180) 2642.22 GETQISDQLTGYGQWEYQANLNK (SEQ ID No: 181) 2984.54 AQNIELVAQYQFDFGLRPSVAYLQSK (SEQ ID No: 182) 3087.49 FGLKGETQISDQLTGYGQWEYQANLNK (SEQ ID No: 183) Lw557 31 863.51 TVYLQIK (SEQ ID No: 184) 1403.71 NTSDKNMLGLAPK + Oxidation (M) (SEQ ID No: 185) 1615.81 FEEAQPVLEDQLAK (SEQ ID No: 186) 1779.83 TQMSETIWLEPSSQK + Oxidation (M) (SEQ ID No: 187) 1875.92 VQTSTQTGNKHQYQTR (SEQ ID No: 188) 2070.10 VNLKFEEAQPVLEDQLAK (SEQ ID No: 189) 2378.15 GYTVTSSPEDAHYWIQANVLK (SEQ ID No: 190) .sup.1Molecular weight as determined by SDS-PAGE. .sup.2The mass of a polypeptide fragment can be converted to m/z value by adding I to the mass. Each mass includes a range of plus or minus 1 Da or plus or minus 300 ppm.

TABLE-US-00002 TABLE 2 Characteristics of polypeptides obtained from Y. pestis. mass of polypeptide approximate fragments molecular resulting weight in from polypeptide kilodaltons trypsin predicted amino acid sequence of the polypeptide designation (kDa).sup.1 digest.sup.2 fragment Lw529 104 643.43 ALISLK (SEQ ID No: 191) 684.36 SIYFR (SEQ ID No: 192) 770.49 ILIGEVK (SEQ ID No: 193) 840.46 NPVARER (SEQ ID No: 194) 898.55 AVQDIILK (SEQ ID No: 195) 961.55 YPLISELK (SEQ ID No: 196) 1136.61 NGIIFSPHPR (SEQ ID No: 197) 1276.63 EAGVQEADFLAK (SEQ ID No: 198) 1292.62 NFEEAVEKAEK (SEQ ID No: 199) 1385.65 VVDESEPFAHEK (SEQ ID No: 200) 1409.76 NGGLNAAIVGQPATK (SEQ ID No: 201) 1421.84 AAALAAADARIPLAK (SEQ ID No: 202) 1497.82 AVTNVAELNELVAR (SEQ ID No: 203) 1566.75 QTAFSQYDRPQAR (SEQ ID No: 204) 1678.89 LLKEFLPASYNEGAK? (SEQ ID No: 205) 1683.82 YAEIADHLGLSAPGDR (SEQ ID No: 206) 1725.93 GSLPIALEEVATDGAKR (SEQ ID No: 207) 1872.90 EYANFSQEQVDKIFR (SEQ ID No: 208) 1990.91 NHFASEYIYNAYKDEK (SEQ ID No: 209) 2020.06 ILINTPASQGGIGDLYNFK (SEQ ID No: 210) 2182.01 EYVEEFDREEEVAAATAPK (SEQ ID No: 211) 2584.21 YNANDNPTKQTAFSQYDRPQAR (SEQ ID No: 212) 2842.48 AAYSSGKPAIGVGAGNTPVVVDETADIKR (SEQ ID No: 213) Lw530 99 1190.64 ILFYTGVNHK (SEQ ID No: 214) 1513.80 YRNIGISAHIDAGK (SEQ ID No: 215) 1590.77 HSDDKEPFSALAFK (SEQ ID No: 216) 1596.83 IATDPFVGNLTFFR (SEQ ID No: 217) 1636.82 YLGGEELTEEEIKK (SEQ ID No: 218) 1670.86 MEFPEPVISVAVEPK (SEQ ID No: 219) 1713.93 EFIPAVDKGIQEQLK (SEQ ID No: 220) 1718.96 LGANPVPLQLAIGAEEK (SEQ ID No: 221) 1750.91 VYSGIVNSGDTVLNSVK (SEQ ID No: 222) 1819.92 EFNVEANVGKPQVAYR (SEQ ID No: 223) 1863.01 EEIKEVHAGDIAAAIGLK (SEQ ID No: 224) 1966.91 LHYGSYHDVDSSELAFK (SEQ ID No: 225) 2122.10 VYSGIVNSGDTVLNSVKSQR (SEQ ID No: 226) Lw531 94 961.44 NRDEWSR (SEQ ID No: 227) 1167.49 YEYGMFSQK +Oxidation (M) (SEQ ID No: 228) 1257.64 VSVIDENNGRR (SEQ ID No: 229) 1371.63 VLYPDDSTYSGR (SEQ ID No: 230) 1383.64 EENDPGLGNGGLGR (SEQ ID No: 231) 1408.71 IIDAPDNNWVPR (SEQ ID No: 232) 1520.82 NLDYPSFLLALQK (SEQ ID No: 233) 1668.86 EYADEIWHIKPIR (SEQ ID No: 234) 1685.79 SYVDTQEQVDALYR (SEQ ID No: 235) 1713.78 GYGIRYEYGMFSQK +Oxidation (M) (SEQ ID No: 236) 1716.81 TLLNIANMGYFSSDR +Oxidation (M) (SEQ ID No: 237) 1796.92 TSPFSYTSPVVSVDALK (SEQ ID No: 238) 1832.92 LVEEQYPDDKELLSR (SEQ ID No: 239) 1844.91 KTLLNIANMGYFSSDR +Oxidation (M) (SEQ ID No: 240) 2218.12 IAIHLNDTHPVLSIPEMMR +2 Oxidation (M) (SEQ ID No: 241) 2426.09 FNQGDYFAAVEDKNHSENVSR (SEQ ID No: 242) Lw532 88 888.51 YIQAAVPK (SEQ ID No: 243) 926.46 FNINYTR (SEQ ID No: 244) 945.53 SGFLIPNAK (SEQ ID No: 245) 960.54 IGFNIELR (SEQ ID No: 246) 1171.60 AQYLYVPYR (SEQ ID No: 247) 1176.57 GLQWQNEFR (SEQ ID No: 248) 1289.64 ITGWNAQGQTSK (SEQ ID No: 249) 1332.67 RGLQWQNEFR (SEQ ID No: 250) 1357.66 EEQVVEVWNAR (SEQ ID No: 251) 1403.74 IASANQVSTGLTSR (SEQ ID No: 252) 1418.68 FTSVNPTNPEASR (SEQ ID No: 253) 1507.73 IYTGPDGTDKNATR (SEQ ID No: 254) 1578.78 FNVSVGQIYYFSR (SEQ ID No: 255) 1672.80 QFQVFTAAGNSNAYR (SEQ ID No: 256) 1735.83 TVTATGDVNYDDPQIK (SEQ ID No: 257) 2400.17 LLATHYQQDIPASFADNASNPK (SEQ ID No: 258) 2665.28 VYNPDYQQGISQVGTTASWPIADR (SEQ ID No: 259) Lw533 77 686.37 DIGNIR (SEQ ID No: 260) 784.49 RIEIVR (SEQ ID No: 261) 858.41 VSYFDTK (SEQ ID No: 262) 952.50 AKDYISTR (SEQ ID No: 263) 1140.65 DLPVSILAGTR (SEQ ID No: 264) 1155.66 QGVLTLVDGVR (SEQ ID No: 265) 1170.64 QVPGLTVTGSGR (SEQ ID No: 266) 1197.57 YYNNSAIEPK (SEQ ID No: 267) 1402.71 EQTTEGVKLENR (SEQ ID No: 268) 1408.68 TDDLDGILSFGTR (SEQ ID No: 269) 1482.73 TALFNWDLAYNR (SEQ ID No: 270) 1522.71 EYYTPQGIPQDGR (SEQ ID No: 271) 1550.77 FSSGWLQDEITLR (SEQ ID No: 272) 1617.74 HSTDTMVVTATGNER (SEQ ID No: 273) 1674.78 QEQTPGGATESFPQAK (SEQ ID No: 274) 1745.84 KHSTDTMVVTATGNER (SEQ ID No: 275) 1787.92 GTWQIDSIQSLSANLR (SEQ ID No: 276) 1819.96 IRFSSGWLQDEITLR (SEQ ID No: 277) 1851.87 VDMQAMTTTSVNIDQAK (SEQ ID No: 278) 1940.75 YDNYSGSSDGYADVDADK (SEQ ID No: 279) 2013.02 QGTDTGHLNSTFLDPALVK (SEQ ID No: 280) 2017.97 QSNGFNAPNDETISNVLAK(SEQ ID No: 281) 2056.96 VYSSAATGDHSFGLGASAFGR (SEQ ID No: 282) 2168.01 VSSSTPQAGYGVNDFYVSYK (SEQ ID No: 283) 2169.10 LFIESPASHLLTYGTETYK (SEQ ID No: 284) 2426.25 TRLFIESPASHLLTYGTETYK (SEQ ID No: 285) 2457.00 YDNYSGSSDGYADVDADKWSSR (SEQ ID No: 286) 2828.33 VSSSTPQAGYGVNDFYVSYKGQEAFK (SEQ ID No: 287) Lw534 73 628.39 IEVIR (SEQ ID No: 288) 748.43 GTIFRR (SEQ ID No;289) 909.42 GGYEDTLR (SEQ ID No: 290) 930.51 TGGLDISIR (SEQ ID No: 291) 1291.71 LLDSLALTYGAR (SEQ ID No: 292) 1370.81 LLKNTNIILDSK (SEQ ID No: 293) 1440.70 FTQNYANLSAANK (SEQ ID No: 294) 1478.71 YDNSANQLGTIGAR (SEQ ID No: 295) 1586.83 EAAASISVISQNELR (SEQ ID No: 296) 1604.86 GMPSAYTLILVDGIR (SEQ ID No: 297) 1640.87 LITNASVPQGSGLAGEK (SEQ ID No: 298) 1654.77 YEYQTTFGGHISPR (SEQ ID No: 299) 1705.82 DASRVESSNTGVELSR (SEQ ID No: 300) 1707.83 AYLVWDAQDNWTVK (SEQ ID No: 301) 1757.91 LNWNINEQLSTWLK (SEQ ID No: 302) 1796.97 LITNASVPQGSGLAGEKR (SEQ ID No: 303) 1856.01 IREAAASISVISQNELR (SEQ ID No: 303) 1912.94 INSVSIDNTTSTYTNVGK (SEQ ID No: 304) 2004.03 DVTLNGAVNNLLDKDFTR (SEQ ID No: 305) 2072.02 FSFYSSGPAVEDQLGLSLR (SEQ ID No: 306) 2155.08 NKINSVSIDNTTSTYTNVGK (SEQ ID No: 307) 2301.07 LDFGTWNSSLSYNQTENIGR (SEQ ID No: 308) 2395.11 NYNDLAQALSDVEGVDVNSSTGK (SEQ ID No: 309) 2484.12 AWASSATLEHTFQENTAFGDSSK (SEQ ID No: 310) 2557.36 VVYNNLGSEFKPFSVLNLGVAYK (SEQ ID No: 311) 2557.36 VVYNNLGSEFKPFSVLNLGVAYK (SEQ ID No: 312) 2675.42 TPTLAQLHNGISGVTGQGTITTIGNPK (SEQ ID No: 313) 2983.33 DGIVLANNGDEFAQDAWSLFSEDEWR (SEQ ID No: 314) 3161.51 THIFAVGNGTTTAGDYFTSSQSTAGYVVPGR (SEQ ID No: 315) 3184.52 ITLGNDNRLDFGTWNSSLSYNQTENIGR (SEQ ID No: 316) 3424.79 GGVSTGYKTPTLAQLHNGISGVTGQGTITTIGNPK (SEQ ID No: 317) 3471.62 LEPESSVNTEVGVYYENETGFGANVTLEHNR (SEQ ID No: 318) Lw535 64 713.42 VPFVPR (SEQ ID No: 319) 759.42 TVGINTR (SEQ ID No: 320) 773.40 AATLGDAR (SEQ ID No: 321) 806.41 YGALMPR (SEQ ID No: 322) 919.48 GPQGTLYGK (SEQ ID No: 323) 1023.50 GYIEGGVSSR (SEQ ID No: 324) 1051.53 SINYELGTR (SEQ ID No: 325) 1102.55 ADATGVELEAK (SEQ ID No: 326) 1164.56 DMQLYSGPVR (SEQ ID No: 327) 1186.57 WNQDVQELR (SEQ ID No: 328) 1199.60 TVDMVFGLYR (SEQ ID No: 329) 1281.67 TVGINTRIDFF (SEQ ID No: 330) 1394.68 YGAGSSVNGVIDTR (SEQ ID No: 331) 1444.73 ADATGVELEAKWR (SEQ ID No: 332) 1479.70 ATQDAYVGWNDIK (SEQ ID No: 333) 1545.80 TFPSGSLIVNMPQR (SEQ ID No: 334) 1667.72 SEFTNDSELYHGNR (SEQ ID No: 335) 1692.82 ATQDAYVGWNDIKGR (SEQ ID No: 336) 1730.85 FAPGWSWDINGNVIR (SEQ ID No: 337) 1789.81 LAPDDQPWEMGFAASR (SEQ ID No: 338) 1904.85 TYGYMNGSSAVAQVNMGR (SEQ ID No: 339) 1968.90 ECTRATQDAYVGWNDIK (SEQ ID No: 340) 1981.02 SAQGGIINIVTQQPDSTPR (SEQ ID No: 341) 2009.89 SSTQYHGSMLGNPFGDQGK (SEQ ID No: 342) 2027.02 LAVNLVGPHYFDGDNQLR (SEQ ID No: 343) 2058.99 YETADVTLQAATFYTHTK (SEQ ID No: 344) 2162.17 FNLSGPIQDGLLYGSVTLLR (SEQ ID No: 345) 2363.13 SEFTNDSELYHGNRVPFVPR (SEQ ID No: 346) 2377.30 SKFNLSGPIQDGLLYGSVTLLR (SEQ ID No: 347) 2819.49 VAQGYKPSGYNIVPTAGLDAKPFVAEK (SEQ ID No: 348) 2929.46 SASANNVSSTVVSAPELSDAGVTASDKLPR (SEQ ID No: 349) Lw536 60 1010.51 VEDALHATR (SEQ ID No: 350) 1186.65 VAAVKAPGFGDR (SEQ ID No: 351) 1230.66 TTLEDLGQAKR (SEQ ID No: 352) 1237.65 ARVEDALHATR (SEQ ID No: 353) 1290.65 VGAATEVEMKEK (SEQ ID No: 354) 1566.87 AAVEEGVVAGGGVALIR (SEQ ID No: 355) 1604.88 NVVLDKSFGSPTITK (SEQ ID No: 356) 1620.85 SFGSPTITKDGVSVAR (SEQ ID No: 357) 1668.75 QQIEDATSDYDKEK (SEQ ID No: 358) 2020.03 AAHAIAGLKGDNEDQNVGIK (SEQ ID No: 359) 2396.29 VVINKDTTIIIDGVGDEAAIQGR (SEQ ID No: 360) Lw537 46 872.51 NLSLLSAR (SEQ ID No: 361) 1000.53 QTVTTPRAQ (SEQ ID No: 362) 1179.55 AAADRDAAYEK (SEQ ID No: 363) 1257.63 NNLDNALESLR (SEQ ID No: 364) 1299.71 LSQDLAREQIK (SEQ ID No: 365) 1306.65 DAAYEKINEVR (SEQ ID No: 366) 1324.65 AIDSLSYTEAQK (SEQ ID No: 367) 1367.75 TQRPDAVNNLLK (SEQ ID No: 368) 1394.76 YNYLINQLNIK (SEQ ID No: 369) 1435.73 ASYDTVLAAEVAAR (SEQ ID No: 370) 1608.93 LKTQRPDAVNNLLK (SEQ ID No: 371) 1615.87 FNVGLVAITDVQNAR (SEQ ID No: 372) 1779.96 TILDVLTATTNLYQSK (SEQ ID No: 373) 1951.01 QITGVYYPELASLNVER (SEQ ID No: 374) 1957.97 AIDSLSYTEAQKQSVYR (SEQ ID No: 375) . 2018.98 QAQYNFVGASELLESAHR (SEQ ID No: 376) 2098.10 SPLLPQLGLSAGYTHANGFR (SEQ ID No: 377) 2177.16 QQLADARYNYLINQLNIK (SEQ ID No: 378) 2709.43 INEVRSPLLPQLGLSAGYTHANGFR (SEQ ID No: 379) Lw538 44 775.40 HTPFFK (SEQ ID No: 380) 836.49 EHILLGR (SEQ ID No: 381) 904.49 FAIREGGR (SEQ ID No: 382) 1026.58 AGENVGVLLR (SEQ ID No: 383) 1072.60 GTVVTGRVER (SEQ ID No: 384) 1199.66 EGGRTVGAGVVAK (SEQ ID No: 385) 1231.57 ALEGEAEWEAK (SEQ ID No: 386) 1232.61 GYRPQFYFR (SEQ ID No: 387) 1289.62 DEGGRHTPFFK (SEQ ID No: 388) 1375.63 AFDQIDNAPEEK (SEQ ID No: 389) 1602.76 AFDQIDNAPEEKAR (SEQ ID No: 390) 1613.89 VGEEVEIVGIKDTVK (SEQ ID No: 391) 1709.94 LLDEGRAGENVGVLLR (SEQ ID No: 392) 1772.87 GITINTSHVEYDTPAR (SEQ ID No: 393) 1794.95 TKPHVNVGTIGHVDHGK (SEQ ID No: 394) 1904.95 ELLSAYDFPGDDLPVVR (SEQ ID No: 395) 1977.01 IIELAGYLDSYIPEPER (SEQ ID No: 396) 2000.01 ARGITINTSHVEYDTPAR (SEQ ID No: 397) Lw683 37 690.4064 VGFAGLK (SEQ ID No: 398) 893.4606 ANAYTGGLK (SEQ ID No: 399) 910.4330 GNGMLTYR (SEQ ID No: 400) 1049.5617 RANAYTGGLK (SEQ ID No: 401) 1114.4931 SSDAAFGFADK (SEQ ID No: 402) 1119.4906 NMSTYVDYK (SEQ ID No: 403) 1121.4697 NGSSSETNNGR (SEQ ID No: 404) 1197.5084 NLDGDQSYMR (SEQ ID No: 405) 1262.5567 FADYGSLDYGR (SEQ ID No: 406) 1307.6146 IDGLHYFSDNK (SEQ ID No: 407) 1319.7085 INLLDKNDFTK (SEQ ID No: 408) 1422.6739 TTAQNDLQYGQGK (SEQ ID No: 409) 1436.6976 YVDIGATYFFNK (SEQ ID No: 410) 1490.6022 AENEDGNHDSFTR (SEQ ID No: 411) 1533.8038 GKDIGIYGDQDLLK (SEQ ID No: 412) 1578.7750 TTAQNDLQYGQGKR (SEQ ID No: 413) 2245.1167 NTNFFGLVDGLNFALQYQGK (SEQ ID No: 414) 2367.1131 YDANNVYLAANYTQTYNLTR (SEQ ID No: 415) 2487.1124 IDGLHYFSDNKNLDGDQSYMR (SEQ ID No: 416) 2684.2718 GETQITDQLTGYGQWEYQVNLNK (SEQ ID No: 417)

2979.5242 AHNIEVVAQYQFDFGLRPSVAYLQSK (SEQ ID No: 418) 3292.4764 GVADQNGDGYGMSLSYDLGWGVSASAAMASSLR (SEQ ID No: 419) Lw541 31 1019.58 ALASNILYR (SEQ ID No: 420) 1074.51 SDPGAAFPWK (SEQ ID No: 421) 1202.61 KSDPGAAFPWK (SEQ ID No: 422) 1247.61 IFNLVDENER (SEQ ID NO: 423) 1321.58 MYNIDYNSFR (SEQ ID No: 424) 1403.64 AWHAGVSYWDGR (SEQ ID No: 425) 1786.80 ALYDAGIGAWYDDETK (SEQ ID No: 426) 1990.03 FPDITPVNVVGHSDIAPGR (SEQ ID No: 427) 2090.99 YGYDTSGAVSEVGYNQLIR (SEQ ID No: 428) 2118.12 FPDITPVNVVGHSDIAPGRK (SEQ ID No: 429) Lw542 31 1142.58 SDPGPLFPWK (SEQ ID No: 430) 1298.68 SDPGPLFPWKR (SEQ ID No: 431) 1307.76 AIALQLVPEAQR (SEQ ID No: 432) 1340.64 AWHAGVSSWQGR (SEQ ID No: 433) 1370.68 IPQNGQLDTETR (SEQ ID No: 434) 1578.77 GTYQIDTHYPSVAK (SEQ ID No: 435) 1779.95 GAASVAVIQQALAAYGYK (SEQ ID No: 436) 1789.94 FLVLHYTAVGDAESLR (SEQ ID No: 437) 1953.00 YNISPSDVVAHSDIAPLR (SEQ ID No: 438) 2190.12 NNLNDTSIGIEIVNLGFTEK (SEQ ID No: 439) 2630.38 AIALQLVPEAQRAWHAGVSSWQGR (SEQ ID No: 440) Lw544 20 806.42 LIDGDFK (SEQ ID No: 441) 1113.50 GFEESVDGFK (SEQ ID No: 442) 1209.60 VGTWMLGAGYR (SEQ ID No: 443) 1243.58 FSSIFGQSESR (SEQ ID No: 444) 1258.63 YYSVTAGPVFR (SEQ ID No: 445) 1269.60 RGFEESVDGFK (SEQ ID No: 446) 1356.66 VGTWMLGAGYRF (SEQ ID No: 447) 1789.94 INEYVSLYGLLGAGHGK (SEQ ID No: 448) 2002.92 YEFNNDWGVIGSFAQTR (SEQ ID No: 449) 2988.43 TSLAYGAGLQFNPHPNFVIDASYEYSK (SEQ ID No: 450) .sup.1Molecular weight as determined by SDS-PAGE. .sup.2The mass of a polypeptide fragment can be converted to m/z value by adding 1 to the mass. Each mass includes a range of plus or minus 300 ppm.

In yet another aspect, the present invention further includes polypeptides having similarity with an amino acid sequence. The similarity is referred to as structural similarity and is generally determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and a reference amino acid sequence) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. Reference amino acid sequences are disclosed in Table 3 and Table 4. Two amino acid sequences can be compared using commercially available algorithms. Preferably, two amino acid sequences are compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al., (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identities." Preferably, a candidate amino acid sequence has at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a reference amino acid sequence. Preferably, the molecular weight of the candidate amino acid sequence and the reference amino acid sequence are substantially the same value. Preferably, the molecular weight of the candidate amino acid sequence and the reference amino acid sequence is determined by SDS polyacrylamide gel electrophoresis. A candidate polypeptide can be obtained by growth of a microbe under low metal conditions and the subsequent isolation of a polypeptide by the procedures disclosed herein.

TABLE-US-00003 TABLE 3 NCBI sequence identifier of polypeptide identified by the Molecular weight computer algorithm as having best of reference match to mass fingerprint of polypeptide (kDa).sup.1 reference polypeptide SEQ ID NO: 268 23630568, adhesin YadA 1 83 282049, hemin receptor precursor 2 83 49114, ferrichrome receptor FcuA 3 79 565634, ferrioxamine receptor 4 70 517386, FyuA precursor 5 66 77958488, Outer membrane receptor 6 for ferrienterochelin and colicins 45 23630568, adhesin YadA 7 37 77956419, Outer membrane protein 8 (porin) 31 48605, YlpA protein 9 .sup.1Molecular weight as determined by SDS-PAGE.

TABLE-US-00004 TABLE 4 NCBI sequence identifier of polypeptide identified by the Molecular weight computer algorithm as having best of reference match to mass fingerprint of polypeptide (kDa).sup.1 reference polypeptide SEQ ID NO: 104 22125915, CoA-linked acetaldehyde 10 dehydrogenase 99 51597993, elongation factor G 11 94 15981846, glycogen phosphorylase 12 88 45443416, organic solvent tolerance 13 protein precursor 77 22124457, TonB-dependent outer 14 membrane receptor 73 51595142, putative exogenous ferric 15 siderophore receptor; Iha adhesin 64 22126288, pesticin/yersiniabactin 16 outer membrane receptor 60 51594757, chaperonin GroEL 17 46 22127390, outer membrane channel 18 precursor protein 44 51597992, elongation factor Tu 19 37 77633559, Outer membrane protein 20 (porin) 31 22125738, putative regulator 21 31 22125770, putative regulator 22 20 22125223, outer membrane protein X 23 .sup.1Molecular weight as determined by SDS-PAGE.

Typically, a candidate amino acid sequence having structural similarity to a reference amino acid sequence has immunogenic activity, protective immunogenic activity, seroactive activity, immunoregulatory activity, or a combination thereof.

The polypeptides expressed by a reference microbe and referred to above by molecular weight can be obtained by growth of the reference microbe under low metal conditions and the subsequent isolation of a polypeptide by the processes disclosed herein. A candidate polypeptide is isolatable from a microbe, preferably a gram negative microbe, more preferably, a member of the family Enterobacteriaceae preferably, a member of the genus Yersinia, such as Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis. A candidate polypeptide may also be produced using recombinant, enzymatic, or chemical techniques.

Also provided by the present invention are whole cell preparations of a microbe, where the microbe expresses one or more of the polypeptides of the present invention. The cells present in a whole cell preparation are preferably inactivated such that the cells cannot replicate, but the immunogenic activity of the polypeptides of the present invention expressed by the microbe is maintained. Typically, the cells are killed by exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

A composition of the present invention may include at least one polypeptide described herein, or a number of polypeptides that is an integer greater than 1 (e.g., at least 2, at least 3, at least 4. In some aspects, a composition may include at least 2 metal regulated polypeptides and at least two polypeptides whose expression is not significantly influenced by the presence of a metal. For example, when the polypeptides are isolatable from Y. enterocolitica, a composition can include 2, 3, 4, 5, or more isolated metal regulated polypeptides having molecular weights of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 45 kDa, or any subset or combination thereof, and two isolated polypeptides having a molecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, 28 kDa, or any subset or combination thereof. In another example, when the polypeptides are isolatable from Y. pestis, a composition can include 2, 3, 4, 5, or more isolated metal regulated polypeptides having molecular weights of 254 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 31 kDa, 28 kDa, 20 kDa, or any subset or combination thereof, and two isolated polypeptides having molecular weights of 104 kDa, 99 kDa, 60 kDa, 44 kDa, 46 kDa, 37 kDa, 36 kDa, or any subset or combination thereof. A composition can include polypeptides isolatable from 1 microbe, or can be isolatable from a combination of 2 or more microbes. For instance, a composition can include polypeptides isolatable from 2 or more Yersinia spp., from 2 or more Y. enterocolitica strains, or from a Yersinia spp. and a different microbe that is not a member of the genus Yersinia. The present invention also provides compositions including a whole cell preparation of one or more Yersinia spp.

Optionally, a polypeptide of the present invention can be covalently bound or conjugated to a carrier polypeptide to improve the immunological properties of the polypeptide. Useful carrier polypeptides are known in the art. For example, a polypeptide of the present invention could be coupled to known Yersinia outer membrane immunogens such as the F1 antigen or the V antigen. Likewise, polysaccharide components could be conjugated to the proteins of the present invention to enhance the protective effect of the compositions. The chemical coupling of polypeptides of the present invention can be carried out using known and routine methods. For instance, various homobifunctional and/or heterobifunctional cross-linker reagents such as bis(sulfosuccinimidyl)suberate, bis(diazobenzidine), dimethyl adipimidate, dimethyl pimelimidate, dimethyl superimidate, disuccinimidyl suberate, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide, sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (see, for instance, Harlow and Lane, Antibodies, A Laboratory Manual, generally and Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, N.Y. (1988)).

Preferably, such compositions of the present invention include low concentrations of lipopolysaccharide (LPS). LPS is a component of the outer membrane of most gram negative microbes (see, for instance, Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.) American Society for Microbiology, Washington, D.C., pp. 7-22 (1987), and typically includes polysaccharides (O-specific chain, the outer and inner core) and the lipid A region. The lipid A component of LPS is the most biologically active component of the LPS structure and together induce a wide spectrum of pathophysiological effects in mammals. The most dramatic effects are fever, disseminated intravascular coagulation, complement activation, hypotensive shock, and death. The non-specific immunostimulatory activity of LPS can enhance the formation of a granuloma at the site of administration of compositions that include LPS.

The concentration of LPS can be determined using routine methods known in the art. Such methods typically include measurement of dye binding by LPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189 (1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, for instance, Endotoxins and Their Detection With the Limulus Amebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. (1982)). There are four basic commercially available methods that are typically used with an LAL test: the gel-clot test; the turbidimetric (spectrophotometric) test; the colorimetric test; and the chromogenic test. An example of a gel-clot assay is available under the tradename E-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma Technical Bulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., East Falmouth, Mass.). Typically, assay conditions include contacting the composition with a preparation containing a lysate of the circulating amebocytes of the horseshoe crab, Limulus polyphemus. When exposed to LPS, the lysate increases in opacity as well as viscosity and may gel. About 0.1 milliliter of the composition is added to lysate. Typically, the pH of the composition is between 6 and 8, preferably, between 6.8 and 7.5. The mixture of composition and lysate is incubated for about 1 hour undisturbed at about 37.degree. C. After incubation, the mixture is observed to determine if there was gelation of the mixture. Gelation indicates the presence of endotoxin. To determine the amount of endotoxin present in the composition, dilutions of a standardized solution of endotoxin are made and tested at the same time that the composition is tested. Standardized solutions of endotoxin are commercially available from, for instance, Sigma Chemical (Catalog No. 210-SE), U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503), and Associates of Cape Cod, Inc., (Catalog No. E0005). In general, when a composition of the present invention is prepared by isolating polypeptides from a microbe by a method as described herein (e.g., a method that includes disrupting and solubilizing the cells, and collecting the insoluble polypeptides), the amount of LPS in a composition of the present invention is less than the amount of LPS present in a mixture of the same amount of the microbe that has been disrupted under the same conditions but not solubilized. Typically, the level of LPS in a composition of the present invention is decreased by, in increasing order of preference, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the level of LPS in a composition prepared by disrupting, but not solubilizing, the same microbe.

The compositions of the present invention optionally further include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a diluent, carrier, excipient, salt, etc, that is compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Typically, the composition includes a pharmaceutically acceptable carrier when the composition is used as described herein. The compositions of the present invention may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration, including routes suitable for stimulating an immune response to an antigen. Thus, a composition of the present invention can be administered via known routes including, for example, oral; parental including intradermal, transcutaneous and subcutaneous, intramuscular, intravenous, intraperitoneal, etc., and topically, such as, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous and rectally etc. It is foreseen that a composition can be administered to a mucosal surface, such as by administration to the nasal or respiratory mucosa (e.g. spray or aerosol), to stimulate mucosal immunity, such as production of secretory IgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via a sustained or delayed release implant. Implants suitable for use according to the invention are known and include, for example, those disclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al., (WO 96/01620 (1996)). Implants can be produced at sizes small enough to be administered by aerosol or spray. Implants also include nanospheres and microspheres.

A composition of the present invention may be administered in an amount sufficient to treat certain conditions as described herein. The amount of polypeptides or whole cells present in a composition of the present invention can vary. For instance, the dosage of polypeptides can be between 0.01 micrograms (.mu.g) and 300 mg, typically between 0.1 mg and 10 mg. When the composition is a whole cell preparation, the cells can be present at a concentration of, for instance, 10.sup.2 bacteria/ml, 10.sup.3 bacteria/ml, 10.sup.4 bacteria/ml, 10.sup.5 bacteria/ml, 10.sup.6 bacteria/ml, 10.sup.7 bacteria/ml, 10.sup.8 bacteria/ml, or 10.sup.9 bacteria/ml. For an injectable composition (e.g. subcutaneous, intramuscular, etc.) the polypeptides may be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0-2.0 ml. When the composition is a whole cell preparation, the cells are preferably present in the composition in an amount that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0-2.0 ml. The amount administered will vary depending on various factors including, but not limited to, the specific polypeptides chosen, the weight, physical condition and age of the animal, and the route of administration. Thus, the absolute weight of the polypeptide included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the animal, as well as the method of administration. Such factors can be determined by one of skill in the art. Other examples of dosages suitable for the invention are disclosed in Emery et al., (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. All methods of preparing a composition including a pharmaceutically acceptable carrier include the step of bringing the active compound (e.g., a polypeptide or whole cell of the present invention) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

A composition including a pharmaceutically acceptable carrier can also include an adjuvant. An "adjuvant" refers to an agent that can act in a nonspecific manner to enhance an immune response to a particular antigen, thus potentially reducing the quantity of antigen necessary in any given immunizing composition, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Adjuvants may include, for example, IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins (including, for instance, those available from under the tradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIM and other substances known in the art. It is expected that polypeptides of the present invention will have immunoregulatory activity, and that such polypeptides may be used as adjuvants that directly act as T and/or B cell activators or act on specific cell types that enhance the synthesis of various cytokines or activate intracellular signaling pathways. Such polypeptides are expected to augment the immune response to increase the protective index of the existing composition.

In another embodiment, a composition of the invention including a pharmaceutically acceptable carrier can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that effect immune cells. An immunizing composition can also include other components known in the art such as an antibiotic, a preservative, an anti-oxidant, or a chelating agent.

The present invention also provides methods for obtaining the polypeptides described herein. The polypeptides and whole cells of the present invention are isolatable from a Yersinia spp. Preferred examples include Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis. Microbes useful for obtaining polypeptides of the present invention and making whole cell preparations are readily available. In addition, such microbes are readily isolatable by techniques routine and known in the art. The microbes may be derived from an infected animal as a field isolate, and used to obtain polypeptides and/or whole cell preparations of the present invention, or stored for future use, for example, in a frozen repository at -20.degree. C. to -95.degree. C., in bacteriological media containing 20% glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from a microbe, the microbe can be incubated under low metal conditions. As used herein, the phrase "low metal conditions" refers to an environment, typically bacteriological media, which contains amounts of a free metal that cause a microbe to express or enhance expression of metal regulated polypeptides. As used herein, the phrase "high metal conditions" refers to an environment that contains amounts of a free metal that cause a microbe to either not express one or more of the metal regulated polypeptides described herein at a detectable level, or to decrease expression of such a polypeptide. Metals are those present in the periodic table under Groups 1 through 17 (IUPAC notation; also referred to as Groups I-A, II-A, III-B, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B, III-A, IV-A, V-A, VI-A, and VII-A, respectively, under CAS notation). Preferably, metals are those in Groups 2 through 12, more preferably, Groups 3-12. Even more preferably, the metal is iron, zinc, copper, magnesium, nickel, cobalt, manganese, molybdenum, or selenium, most preferably, iron.

Low metal conditions are generally the result of the addition of a metal chelating compound to a bacteriological medium, or the use of bacteriological media formulated to contain low amounts of a metal. High metal conditions are generally present when a chelator is not present in the medium, a metal is added to the medium, or the combination thereof. Examples of metal chelators include natural and synthetic compounds. Examples of natural compounds include plant phenolic compounds, such as flavenoids. Examples of flavenoids include the copper chelators catechin and naringenin, and the iron chelators myricetin and quercetin. Examples of synthetic copper chelators include, for instance, tetrathiomolybdate, and examples of synthetic zinc chelators include, for instance, N,N,N',N'-Tetrakis(2-pyridylmethyl)-ethylene diamine. Examples of synthetic iron chelators include 2,2'-dipyridyl (also referred to in the art as .alpha.,.alpha.'-bipyridyl), 8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxamine methanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin, biological siderophores, such as, the catecholates and hydroxamates, and citrate. Preferably, 2,2'-dipyridyl is used for the chelation of iron. Typically, 2,2'-dipyridyl is added to the media at a concentration of at least 0.0025 micrograms/milliliter (.mu.g/ml), at least 0.025 .mu.g/ml, or at least 0.25 .mu.g/ml, and generally no greater than 10 .mu.g/ml, no greater than 20 .mu.g/ml, or no greater than 30 .mu.g/ml.

It is expected that a Yersinia spp. with a mutation in a fur gene will result in the constitutive expression of many, if not all, of the iron regulated polypeptides of the present invention. The production of a fur mutation in a Yersinia spp. can be produced using routine methods including, for instance, transposon, chemical, or site-directed mutagenesis useful for generating gene knock-out mutations in gram negative bacteria.

The medium used to incubate the microbe and the volume of media used to incubate the microbe can vary. When a microbe is being evaluated for the ability to produce one or more of the polypeptides described herein, the microbe can be grown in a suitable volume, for instance, 10 milliliters to 1 liter of medium. When a microbe is being grown to obtain polypeptides for use in, for instance, administration to animals, the microbe may be grown in a fermentor to allow the isolation of larger amounts of polypeptides. Methods for growing microbes in a fermentor are routine and known in the art. The conditions used for growing a microbe preferably include a metal chelator, more preferably an iron chelator, for instance 2,2'-dipyridyl, a pH of between 6.5 and 7.5, preferably between 6.9 and 7.1, and a temperature of 37.degree. C.

In some aspects of the invention, a microbe may be harvested after growth. Harvesting includes concentrating the microbe into a smaller volume and suspending in a media different than the growth media. Methods for concentrating a microbe are routine and known in the art, and include, for example, filtration or centrifugation. Typically, the concentrated microbe is suspended in decreasing amounts of buffer. Preferably, the final buffer includes a cation chelator, preferably, ethylenediaminetetraacetic acid (EDTA). An example of a buffer that can be used contains Tris-base (7.3 grams/liter) and EDTA (0.9 grams/liter), at a pH of 8.5. Optionally, the final buffer also minimizes proteolytic degradation. This can be accomplished by having the final buffer at a pH of greater than 8.0, preferably, at least 8.5, and/or including one or more proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride). Optionally and preferably, the concentrated microbe is frozen at -20.degree. C. or below until disrupted.

When the microbe is to be used as a whole cell preparation, the harvested cells may be processed using routine and known methods to inactivate the cells. Alternatively, when a microbe is to be used to prepare polypeptides of the present invention, the microbe may be disrupted using chemical, physical, or mechanical methods routine and known in the art, including, for example, french press, sonication, or homogenization. Preferably, homogenization is used. An example of a suitable device useful for homogenization is a model C500 Avestin Homogenizer, (Avestin Inc, Ottawa Canada). As used herein, "disruption" refers to the breaking up of the cell. Disruption of a microbe can be measured by methods that are routine and known in the art, including, for instance, changes in optical density. Typically, a microbe is subjected to disruption until the percent transmittance is increased by 20% when a 1:100 dilution is measured. The temperature during disruption is typically kept low, preferably at 4.degree. C., to further minimize proteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, an anionic, zwitterionic, nonionic, or cationic detergent. Preferably, the detergent is sarcosine, more preferably, sodium lauroyl sarcosinate. As used herein, the term "solubilize" refers to dissolving cellular materials (e.g., polypeptides, nucleic acids, carbohydrates) into the aqueous phase of the buffer in which the microbe was disrupted, and the formation of aggregates of insoluble cellular materials. The conditions for solubilization preferably result in the aggregation of polypeptides of the present invention into insoluble aggregates that are large enough to allow easy isolation by, for instance, centrifugation.

Significant decreases in LPS are typically observed when the disrupted microbe is solubilized in higher levels of sarcosine, solubilized for longer periods, or the combination thereof. Preferably, the sarcosine is added such that the final ratio of sarcosine to gram weight of disrupted microbe is between 1.0 gram sarcosine per 4.5 grams pellet mass and 6.0 grams sarcosine per 4.5 grams pellet mass, preferably, 4.5 gram sarcosine per 4.5 grams pellet mass. The solubilization of the microbe may be measured by methods that are routine and known in the art, including, for instance, changes in optical density. Typically, the solubilization is allowed to occur for at least 24 hours, preferably, at least 48 hours, more preferably, at least 72 hours, most preferably, at least 96 hours. The temperature during disruption is typically kept low, preferably at 4.degree. C.

The insoluble aggregates that include one or more of the polypeptides of the present invention may be isolated by methods that are routine and known in the art. Preferably, the insoluble aggregates are isolated by centrifugation. Typically, centrifugation of polypeptides that are insoluble in detergents requires centrifugal forces of at least 50,000.times.g, typically 100,000.times.g. The use of such centrifugal forces requires the use of ultracentrifuges, and scale-up to process large volumes of sample is often difficult and not economical with these types of centrifuges. The methods described herein provide for the production of insoluble aggregates large enough to allow the use of significantly lower centrifugal forces (for instance, 46,000.times.g). Methods for processing large volumes at these lower centrifugal forces are available and known in the art. Thus, the insoluble aggregates can be isolated at a significantly lower cost. Examples of suitable devices useful for centrifugation of large volumes include T-1 Sharples, (Alfa Laval Separations, Warminster, Pa.) and Hitachi Himac CC40 high speed centrifuges (Hitachi-Koki Co, Tokyo, Japan).

Optionally and preferably, the sarcosine is removed from the isolated polypeptides. Methods for removing sarcosine from the isolated polypeptides are known in the art, and include, for instance, diafiltration, precipitation, hydrophobic chromatography, ion-exchange chromatography, or affinity chromatography, and ultra filtration and washing the polypeptides in alcohol by diafiltration. After isolation, the polypeptides suspended in buffer and stored at low temperature, for instance, -20.degree. C. or below.

Polypeptides of the present invention may also be obtained from members of the genus Yersinia using methods that are known in the art. The isolation of the polypeptides may be accomplished as described in, for instance, Emery et al., (U.S. Pat. No. 5,830,479) and Emery et al., (U.S. Patent Application US 20030036639 A1).

In those aspects of the present invention where a whole cell preparation is to be made, methods known in the art can be used. For instance, after growth a microbe can be killed with the addition of an agent such as glutaraldehyde, formalin, or formaldehyde, at a concentration sufficient to inactivate the cells in the culture. For instance, formalin can be added at a concentration of 3% (vol:vol). After a period of time sufficient to inactivate the cells, the cells can be harvested by, for instance, diafiltration and/or centrifugation, and washed.

An aspect of the present invention is further directed to methods of using the compositions of the present invention. The methods include administering to an animal an effective amount of a composition of the present invention. The animal can be, for instance, avian (including, for instance, chickens or turkeys), bovine (including, for instance, cattle), caprine (including, for instance, goats), ovine (including, for instance, sheep), porcine (including, for instance, swine), bison (including, for instance, buffalo), a companion animal (including, for instance, cats, dogs, and horses), members of the family Cervidae (including, for instance, deer, elk, moose, caribou, and reindeer), piscine (including, for instance, salmon or trout), crustacean (including, for instance, lobster, crab, or shrimp), members of the family Muridae (including, for instance, rats or mice), or human.

In some aspects, the methods may further include additional administrations (e.g., one or more booster administrations) of the composition to the animal to enhance or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually. Without intending to be limited by theory, it is expected that in some aspects of the present invention annual boosters will not be necessary, as an animal will be challenged in the field by exposure to microbes expressing polypeptides present in the compositions having epitopes that are identical to or structurally related to epitopes present on polypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for making antibody, such as inducing the production of antibody in an animal, or by recombinant techniques. The antibody produced includes antibody that specifically binds at least one polypeptide present in the composition. In this aspect of the invention, an "effective amount" is an amount effective to result in the production of antibody in the animal. Methods for determining whether an animal has produced antibodies that specifically bind polypeptides present in a composition of the present invention can be determined as described herein. The present invention further includes antibody that specifically bind to a polypeptide of the present invention, and compositions including such antibodies.

The method may be used to produce antibody that specifically binds polypeptides expressed by a microbe other than the microbe from which the polypeptides of the composition were isolated. As used herein, an antibody that can "specifically bind" a polypeptide is an antibody that interacts with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. At least some of the polypeptides present in the compositions of the present invention typically include epitopes that are conserved in the polypeptides of different species of microbes. Accordingly, antibody produced using a composition derived from one microbe is expected to bind to polypeptides expressed by other microbes and provide broad spectrum protection against gram negative organisms. Examples of gram negative microbes to which the antibody may specifically bind are enteropathogens, for instance, members of the family Enterobacteriaceae, preferably, members of the genus Yersinia.

The present invention is also directed to the use of such antibody to target a microbe expressing a polypeptide of the present invention or a polypeptide having an epitope structurally related to an epitope present on a polypeptide of the present invention. A compound can be covalently bound to an antibody, where the compound can be, for instance, a toxin. Likewise, such compounds can be covalently bound to a bacterial siderophore, such as yersiniabactin, to target the microbe. The chemical coupling or conjugation of an antibody of the present invention or a portion thereof (such as an Fab fragment) can be carried out using known and routine methods.

In one aspect the invention is also directed to treating an infection in an animal caused by a gram negative microbe, preferably by a member of the genus Yersinia. As used herein, the term "infection" refers to the presence of a gram negative microbe, preferably, a member of the genus Yersinia, in an animal's body, which may or may not be clinically apparent. An animal with an infection by member of the genus Yersinia that is not clinically apparent is often referred to as an asymptomatic carrier. The method includes administering an effective amount of the composition of the present invention to an animal having an infection caused by a member of the genus Yersinia, and determining whether the Yersinia spp. causing the infection has decreased. Methods for determining whether an infection is caused by a member of the genus Yersinia are routine and known in the art.

In another aspect, the present invention is directed to methods for treating one or more symptoms of certain conditions in animals such as sheep, cattle, goats, pigs, dogs, birds, rodents and deer that may be caused by infection by a member of the genus Yersinia. Examples of conditions caused by Yersinia spp. infections include, for instance, diarrhea or enteritis in bovine, ovine, and porcine animals and humans, plague-like illnesses in domestic cats and humans, abortion in cattle and sheep, epididymitis-orchitis in rams, and multiple abscess formation in sheep. Yet another aspect of the present invention is directed at treating cold water diseases of fish such as enteric red mouth disease in juvenile fish, particularly in intensive aquaculture of trout and salmon. Treatment of symptoms associated with these conditions can be prophylactic or, alternatively, can be initiated after the development of a condition described herein. As used herein, the term "symptom" refers to objective evidence in a subject of a condition caused by infection by a member of the genus Yersinia spp. Symptoms associated with conditions referred to herein and the evaluation of such symptoms are routine and known in the art. Treatment that is prophylactic, for instance, initiated before a subject manifests symptoms of a condition caused by a microbe, is referred to herein as treatment of a subject that is "at risk" of developing the condition. Typically, an animal "at risk" of developing a condition is an animal present in an area where the condition has been diagnosed and/or is likely to be exposed to a Yersinia spp. causing the condition. Accordingly, administration of a composition can be performed before, during, or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms. In this aspect of the invention, an "effective amount" is an amount effective to prevent the manifestation of symptoms of a disease, decrease the severity of the symptoms of a disease, and/or completely remove the symptoms.

The present invention is also directed to decreasing the colonization by gram negative bacteria, for instance blocking the attachment sites by gram negative bacteria, to tissues of the skeletal system (for instance, bones, cartilage, tendons and ligaments), muscular system, (for instance, skeletal and smooth muscles), circulatory system (for instance, heart, blood vessels, capillaries and blood), nervous system (for instance, brain, spinal cord, and peripheral nerves), respiratory system (for instance, nose, trachea lungs, bronchi, bronchioceles, alveoli), digestive system (for instance, mouth, salivary glands oesophagus liver stomach large and small intestine), excretory system (for instance, kidneys, ureters, bladder and urethra), endocrine system (for instance, hypothalamus, pituitary, thyroid, pancreas and adrenal glands), reproductive system (for instance, ovaries, oviduct, uterus, vagina, mammary glands, testes, and seminal vesicles), lymphatic/immune systems (for instance, lymph, lymph nodes and vessels, mononuclear or white blood cells, such as macrophages, neutrophils, monocytes, eosinophils, basophils, lymphocytes t- and b-cells), and specific cell lineages (for instance, precursor cells, epitheial cells, stem cells), and the like. Preferably, the gram negative bacteria is a member of the genus Yersinia. The method includes administering an effective amount of a composition of the present invention to an animal colonized by, or at risk of being colonized by a member of the genus Yersinia. In this aspect of the invention, an "effective amount" is an amount effective to decrease colonization of the animal by the microbe. Methods for evaluating the colonization of an animal by a microbe are routine and known in the art. For instance, colonization of an animal's intestinal tract by a microbe can be deter mined by measuring the presence of the microbe in the animal's feces. It is expected that decreasing the colonization of an animal by a microbe will reduce transmission of the microbe to humans.

A composition of the invention can be used to provide for active or passive immunization against bacterial infection. Generally, the composition can be administered to an animal to provide active immunization. However, the composition can also be used to induce production of immune products, such as antibodies, which can be collected from the producing animal and administered to another animal to provide passive immunity. Immune components, such as antibodies, can be collected to prepare antibody compositions from serum, plasma, blood, colostrum, etc. for passive immunization therapies. Antibody compositions comprising monoclonal antibodies and/or anti-idiotypes can also be prepared using known methods. Such antibody compositions include chimeric antibodies and humanized antibodies. Chimeric antibodies include human-derived constant regions of both heavy and light chains and murine-derived variable regions that are antigen-specific (Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81(21):6851-5; LoBuglio et al., Proc. Natl. Acad. Sci. USA, 1989, 86(16:4220-4; Boulianne et al., Nature, 1984, 312(5995):643-6). Humanized antibodies substitute the murine constant and framework (FR) (of the variable region) with the human counterparts (Jones et al., Nature, 1986, 321(6069):522-5; Riechmann et al., Nature, 1988, 332(6162):323-7; Verhoeyen et al., Science, 1988, 239(4847):1534-6; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86(24):10029-33; Daugherty et al., Nucleic Acids Res., 1991, 19(9): 2471-6). Alternatively, certain mouse strains can be used that have been genetically engineered to produce antibodies that are almost completely of human origin; following immunization the B cells of these mice are harvested and immortalized for the production of human monoclonal antibodies (Bruggeman and Taussig, Curr. Opin. Biotechnol., 1997, 8(4):455-8; Lonberg and Huszar, Int. Rev. Immunol., 1995; 13(1):65-93; Lonberg et al., Nature, 1994, 368:856-9; Taylor et al., Nucleic Acids Res., 1992, 20:6287-95). Passive antibody compositions and fragments thereof, e.g., scFv, Fab, F(ab').sub.2 or Fv or other modified forms thereof, may be administered to a recipient in the form of serum, plasma, blood, colostrum, and the like. However, the antibodies may also be isolated from serum, plasma, blood, colostrum, and the like, using known methods for later use in a concentrated or reconstituted form such as, for instance, lavage solutions, impregnated dressings and/or topical agents and the like. Passive immunizing preparations may be particularly advantageous for treatment of acute systemic illness, or passive immunization of young animals that failed to receive adequate levels of passive immunity through maternal colostrum. Antibodies useful for passive immunization may also be useful to conjugate to various drugs or antibiotics that could be directly targeted to bacteria expressing during a systemic or localized infection a polypeptide of the present invention or a polypeptide having an epitope structurally related to an epitope present on a polypeptide of the present invention.

Animal models, in particular mouse models, are available for experimentally evaluating the compositions of the present invention (see, for instance, Alpar, H. O., et al., Adv. Drug Deliv. Rev., 51, 173-201, (2001), Brem, D., et al., Microbiology, 147, 1115-1127, (2001), Carter, P. B. and F. M. Collins, Infect. Immun., 9, 851-857, (1974), Collyn, F., et al., Infect. Immun., 72, 4784-9470, (2004), Di Genaro, M. S., et al., Microbiol. Immunol., 42, 781-788, (1998), Grosfeld, H., et al., Infect Immun, 71, 374-383, (2003), Jones, S. M., et al., Vaccine, 19, 358-366, (2001), Karlyshev, A. V., et al., Infect Immun, 69, 7810-7819, (2001), Leary, S. E., et al., Microb Pathog, 23, 167-179, (1997), Noll, A., et al., Eur J Immunol, 29, 986-996, (1999), Pelludat, C., et al., Infect Immun, 70, 1832-1841, (2002), Sabhnani, L., et al., FEMS Immunol Med Microbiol, 38, 215-29, (2003), and Williamson, E. D., et al., Vaccine, 19, 566-571, (2000)). These mouse models are commonly accepted models for the study of human disease caused by members of the genus Yersinia, and additionally have served as accepted models in the development and initial testing of vaccines aimed at preventing human illnesses by Yersinia spp.

Another aspect of the present invention provides methods for detecting antibody that specifically binds polypeptides of the present invention. These methods are useful in, for instance, detecting whether an animal has antibody that specifically bind polypeptides of the present invention, and diagnosing whether an animal may have a condition caused by a microbe expressing polypeptides described herein, or expressing polypeptides that share epitopes with the polypeptides described herein. Such diagnostic systems may be in kit form. The methods include contacting an antibody with a preparation that includes polypeptides of the present invention to result in a mixture. The antibody may be present in a biological sample, for instance, blood, milk, or colostrum. The method further includes incubating the mixture under conditions to allow the antibody to specifically bind the polypeptide to form a polypeptide:antibody complex. As used herein, the term "polypeptide:antibody complex" refers to the complex that results when an antibody specifically binds to a polypeptide. The preparation that includes the polypeptides of the present invention may also include reagents, for instance a buffer, that provide conditions appropriate for the formation of the polypeptide:antibody complex. The polypeptide:antibody complex is then detected. The detection of antibodies is known in the art and can include, for instance, immunofluorescence and peroxidase. The methods for detecting the presence of antibodies that specifically bind to polypeptides of the present invention can be used in various formats that have been used to detect antibody, including radioimmunoassay and enzyme-linked immunosorbent assay.

The present invention also provides a kit for detecting antibody that specifically binds polypeptides of the present invention. The antibody detected may be obtained from an animal suspected to have an infection caused by a gram negative microbe, more preferably, a member of the family Enterobacteriaceae preferably, a member of the genus Yersinia, such as Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis. The kit includes at least one of the polypeptides of the present invention, or a number of polypeptides that is an integer greater than 1 (e.g., at least 2, at least 3, etc.), in a suitable packaging material in an amount sufficient for at least one assay. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. For instance, a kit may also include a reagent to permit detection of an antibody that specifically binds to a polypeptide of the present invention, such as a detectably labeled secondary antibody designed to specifically bind to an antibody obtained from an animal. Instructions for use of the packaged polypeptides are also typically included. As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by well known methods, generally to provide a sterile, contaminant-free environment. The packaging material may have a label which indicates that the polypeptides can be used for detecting antibody that specifically binds polypeptides of the present invention. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to detect the antibody. As used herein, the term "package" refers to a container such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the polypeptides, and other reagents, for instance a secondary antibody. Thus, for example, a package can be a microtiter plate well to which microgram quantities of polypeptides have been affixed. A package can also contain a secondary antibody. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

Production and Isolation of Metal Regulated Proteins

The compositions used in the following examples were prepared using the proteins derived from Y. enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ (obtained from R. D. Perry, University of Kentucky). The two strains were each inoculated from frozen stocks into 25 ml tryptic soy broth (TSB) containing 160 .mu.M 2,2-diprydyl or 300 .mu.M FeCl.sub.3, and incubated at 37.degree. C. while shaking at 400 rpm. Following 12 hours of incubation, 5 ml of each culture was transferred into 500 ml of pre-incubated (37.degree. C.) media containing 160 .mu.M 2,2-diprydyl or 300 .mu.M FeCl.sub.3 and incubated at 37.degree. C. while stirring at 100 rpm. After 8 hours of incubation, the cultures were centrifuged at 10,000.times.g for 20 minutes. The bacterial pellets were resuspended in 100 ml of sterile physiological saline and centrifuged at 10,000.times.g for 10 minutes to remove any contaminating media proteins. The bacterial pellets were then resuspended in 40 ml of Tris-buffered saline pH 7.2 (TBS) and disrupted by sonication for 1.5 minutes at 4.degree. C. using a Branson 450 equipped with a half inch disruption horn (Branson, Danbury Conn.). The disrupted bacterial suspensions were clarified by centrifugation at 32,000.times.g for 12 minutes. The supernatants were collected and solubilized by the addition of sodium lauroyl sarcosinate (4% vol/vol) at 4.degree. C. for 24 hours. The detergent-insoluble protein-enriched fractions were collected by centrifugation at 32,000.times.g for 2.5 hours at 4.degree. C. The protein pellets were resuspended in 200 .mu.l Tris-buffer (pH 7.2) and stored at -90.degree. C. A sample of each extract was resolved on a 10% SDS-PAGE gel per standard methods and visualized by Coomassie Blue staining (FIG. 3).

Example 2

Preparation of the Immunizing Compositions Derived from Y. enterocolitica

The proteins made from Y. enterocolitica as described in Example 1 were used to prepare a composition for administration to animals. The composition contained polypeptides having molecular weights of 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa. The polypeptides having molecular weights of 83 kDa, 70 kDa, and 66 kDa were expressed only under iron limited conditions, and the expression of polypeptides having molecular weights of 268 kDa, 79 kDa, and 45 kDa was enhanced under iron limited conditions.

A stock vaccine was prepared from the composition by emulsifying the aqueous protein suspension (500 .mu.g total protein/ml) into the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) using an IKA Ultra Turrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio). The vaccine was administered to mice to give a final dose of 50 .mu.g total protein in a 0.1 ml injectable volume with an adjuvant concentration of 22.5% vol/vol. A placebo was prepared by replacing the antigen with physiological saline in the above formulation and emulsifying the suspension into EMULSIGEN to give an adjuvant concentration of 22.5%.

Example 3

Preparation of Challenge Organism

When used as a challenge, the Y. enterocolitica ATCC strain 27729 was prepared as follows. Briefly, the isolate from a frozen stock was streaked onto a blood agar plate and incubated at 37.degree. C. for 18 hours. A single colony was subcultured into 50 ml Tryptic Soy Broth (Difco) containing 25 .mu.g/ml 2,2' dipyridyl. The culture was incubated at 37.degree. C. for 6 hours while rotating at 200 rpm, at which point the culture was centrifuged at 10,000.times.g for 10 minutes at 4.degree. C. to pellet the bacteria. The bacterial pellet was washed twice by centrifugation in physiological saline at 4.degree. C. The final pellet was resuspended in 25 ml of physiological saline and used for challenge. Just prior to challenge, 1 ml of the above bacterial suspension was serially diluted ten fold to enumerate the number of CFU/mouse dose.

Example 4

Mouse Vaccination and Challenge Study to Evaluate Protection Against Intravenous Challenge

The efficacy of the Y. enterocolitica composition was evaluated using a live virulent challenge in mice. Twenty CF-1 mice (Harlan Breeding Laboratories, Indianapolis, Ind.) were divided into two groups of 10 mice per group. Mice in the control group were vaccinated with the placebo, while mice in the second group were immunized with 50 .mu.g of the composition obtained as described in Example 1. Immunizations of 0.1 cc were administered intraperitoneally two times at 14 day intervals. Fourteen days after the second vaccination, a challenge dose of strain 27729 (9.4.times.10.sup.4 CFU/mouse) was administered to all mice in the lateral tail vein. Mortality was recorded for 7 days following challenge.

Of the 10 placebo-vaccinated mice, 10 (100%) died within 168 hours of challenge, while none of the vaccinated mice died within the same time period. Furthermore, all of the vaccinated mice survived for the remainder of the study, which was terminated at 20 days post-challenge. A Kaplan-Meier survival analysis and logrank test (see FIG. 1) indicated that immunization provided statistically significant (p<0.0001-) protection against challenge. These results suggest that proteins from Y. enterocolitica grown under iron-restricted conditions constitute effective antigens in the intravenous mouse model of infection.

Example 5

Western Blot Analysis of Y. enterocolitica Proteins with Hyperimmunized and Convalescent Mouse Polyclonal Serum

Western blot analysis was used to evaluate the immuno-reactive proteins derived from Y. enterocolitica against hyperimmunized mouse polyclonal serum and convalescent sera. Hyperimmunizzed mouse polyclonal serum was obtained after vaccinating with the composition described in example 2, and convalescent sera was obtained from vaccinated/challenged mice that survived the trial descrived in example 4. The composition contained polypeptides having molecular weights of 268 kDa, 92 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 52 kDa, 41 kDa, 38 kDa, 37 kDa, 31 kDa, 28 kDa, and two proteins having molecular weights of 83 kDa. The polypeptides having molecular weights of 83 kDa, 70 kDa, and 66 kDa were expressed only under iron limited conditions.

To obtain hyper-immunized serum, mice were immunized two times at 14 day intervals as described in Example 4. The hyperimmunized polyclonal serum was collected from mice 14 days following the second immunization. Convalescent serum derived from vaccinated/challenged mice was obtained 14 days after challenge. The proteins derived from Y. enterocolitica strain 27729 were first size fractionated on SDS-PAGE (4% stacker/10% resolving gel) using 30 ug total protein as described in example 1. Band migration was visualized using broad range kaleidoscope standards (BioRad) to aid in the electroblot transfer while biotinylated broad range standards were used as molecular weight references on the blot. For western blot analysis, proteins were electroblotted from the SDS-PAGE gel onto trans-blot nitrocellulose membranes (BioRad) overnight, at 4.degree. C. at 50 Volts, in Towbin buffer (25 mM Tris, 192 mM glycine, and 20% methanol) using a BioRad Trans-Blot transfer cell. The nitrocellulose membrane was blocked by standard methods using 3.0% fish gelatin (BioRad). The hyperimmunized polyclonal serum and convalescent sera was diluted 1/25000 in Tris-buffered saline containing 1.0% fish gelatin, 0.05% tween 20 and 0.2% sodium azide (antibody buffer). The nitrocellulose membrane was incubated with the primary antibody solution overnight. The membrane was then washed two times in Tris-Buffered Saline containing 0.05% tween 20 (TTBS) and transferred to antibody buffer containing a 1/10,000 dilution of goat anti-mouse antibody conjugated to alkaline phosphatase (BioRad) and a 1/3,000 dilution of avidin conjugated to alkaline phosphatase (BioRad). The membrane was incubated at 37.degree. C. for 2 hours on a shaker, and subsequently washed in TTBS four times to remove unbound conjugate. The blot was resolved, for 30 minutes at 37.degree. C. on a shaker, in substrate solution containing alkaline phosphate color reagent A and B in 1.times.AP color development buffer (BioRad).

Western blot analysis was used as a tool to potentially identify proteins derived from the composition as described in example 1 as immuno-reactive with antibodies derived from the hyperimmunized and/or convalescent sera. Western blot analysis revealed a number of immuno-reactive proteins. The hyperimmunized sera contained antibodies that reacted with proteins at the 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 52 kDa, 41 kDa, 38 kDa, 37 kDa, 31 kDa and 28 kDa. Similarly, the convalescent sera showed identical banding patterns at the 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 52 kDa, 41 kDa, 38 kDa, 37 kDa, 31 kDa and 28 kDa. In addition, three immuno-reactive proteins were seen at the 52 kDa, 40 kDa and 20 kDa regions that were not seen on the SDS-PAGE gel initially, nor were they seen in the western blot analysis using the hyperimmunized sera. It is interesting to speculate that these three proteins were at too low of concentration to be visualized on the SDS-PAGE gel, but may be highly immunogenic resulting in greater band intensity after priming the immune system that resulted in an enhanced band intensity of these proteins after challenge.

The Western Blot analysis of the vaccine composition revealed differences in band intensities of the immuno-reactive proteins between both the hyperimmunized and convalescent sera. These differences could be the result of different immunogenic properties of individual proteins and how the immune system recognizes each individual protein within the composition. In addition, the amount and ratio of proteins within the composition can also influence the immunological status of each protein which can influence the immunological response of the animal to individual proteins within the composition. Nevertheless, each protein within the composition reacted immunologically as examined by Western Blot Analysis, thus the immunological response of the mouse upon vaccination, recognized and responded mounting an antibody response to each individual protein within the composition. Taken together, the results as described in example 4 illustrate that the protein composition was extremely efficacious providing a 100% protection in challenged mice compared to the non-vaccinated mice having 100% mortality.

Example 6

Western Blot Analysis of Y. pestis Proteins with Hyperimmunized Serum Prepared Against Proteins of Y. enterocolitica

Western blot analysis was used to evaluate the immuno-reactive proteins derived from Y. pestis against hyperimmunized sera prepared against the composition derived from Y. enterocolitica as described in example 5. The composition contained polypeptides having molecular weights of 254 kDa, 104 kDa, 99 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 60 kDa, 46 kDa, 44 kDa, 37 kDa 36 kDa, 31 kDa 28 kDa and 20 kDa. The polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa were expressed only under iron limited conditions. The proteins derived from Y. pestis strain KIM6+ was first size fractionated on SDS-PAGE (4% stacker/10% resolving gel) as previously described in example 5 using 30 ug total protein. Western blot analysis was run under identical conditions as described in example 5 except for the following modification; the convalescent sera was not tested against the membrane proteins of Y. pestis. The results showed proteins at approximately the 254 kDa, 94 kDa, 88 kDa, 46 kDa, 44 kDa, 37 kDa, 36 kDa and 20 kDa regions to be immuno-reactive with antibodies derived from the hyperimmunized serum prepared against membrane proteins of Y. enterocolitica.

Example 7

Mouse Vaccination and Challenge Study to Evaluate Protection Against Intravenous and Pneumonic Y. pestis Challenge

Eighty-eight female Swiss-Webster (Harlan Laboratories) weighing 16-22 grams are equally distributed into 4 groups (22 mice/group), designated 1 through 4. The animals are housed in a HEPA-filtered, micro-vent positive air supply animal caging system (BSL3 facility). Food and water are supplied ad libitum.

Proteins from Y. pestis strain KIM6+ are prepared as described above in example 1, and formulated as a vaccine using aluminum hydroxide as the adjuvant (Rehydagel-HPA, Rheis N.J.) at a final concentration of 20% vol/vol and 500 .mu.g total protein/ml. The placebo is prepared by replacing the antigen with PBS while maintaining the same adjuvant concentration. Mice in Groups 1 and 3 are vaccinated intraperitoneally two times at 14 day intervals with 0.1 ml of vaccine containing 50 .mu.g total protein, while mice in Groups 2 and 4 are immunized with the placebo by an identical schedule.

Y. pestis strain CO92 is used for challenge, and is prepared in a BSL3 containment facility. Fourteen days after the second vaccination, mice in Groups 1 and 2 are challenged intravenously in the lateral tail vein with 0.1 ml strain CO92 (10.sup.3 CFU or approximately 100 LD.sub.50 per mouse). Mice in groups 3 and 4 are subjected to an aerosolized challenge dose of Y. pestis C092 diluted in physiological saline to achieve an approximate concentration of 100 LD.sub.50 CFU per mouse for 30 minutes in an airtight chamber. The aerosolized LD.sub.50 for strain C092 in Swiss Webster mice is determined by small pilot studies prior to the proposed challenge experiments. Mortality is recorded for 21 days after challenge.

Example 8

Fish Vaccination and Challenge Study to Evaluate Protection Against Y. ruckeri Challenge

Two groups of 20 rainbow trout, designated as groups 1 and 2 weighing approximately 2 grams are maintained in two separate 60 liter tanks at a temperature of 18.degree. C. Fish are fed twice daily with a commercial trout feed (Ziegler Brothers, Gardners, Pa.). Fish in group 1 are vaccinated with a composition derived from Y. ruckeri using the same method as described in example 1. The extracted proteins derived from Y. ruckeri are used to prepare a vaccine composition for administration to fish. A stock vaccine is prepared from the composition by emulsifying the aqueous protein suspension into a water-in-oil emulsion containing Drakeol 6 mineral oil and Arlacel A as an emulsifier. The vaccine is administered intraperitoneally to give a final dose of 25 ug total protein in a 0.1 cc injectable volume using 0.1 cc. A placebo is prepared by replacing the antigen with physiological saline in the above formulation and is given to the fish in group 2 (controls). Fish are given a second vaccination 28 days after the first vaccination. Fourteen days after the second vaccination all fish are intraperitoneally challenged.

A virulent isolate of Y. ruckeri is used for challenge. The challenge isolate is cultured in Tryticase Soy Broth (TSB) containing 160 .mu.M 2,2-diprydyl and grown for 12 hours of incubation at 37.degree. C. The culture is washed once in physiological saline by centrifugation at 10,000.times.g and resuspended in saline. The culture is adjusted to 5.0.times.10.sup.7 CFU per ml. Each trout is intraperitoneally inoculated with 0.1 cc of the corresponding bacteria at a final challenge dose of 5.0.times.10.sup.6 CFU. Mortality was recorded daily for 14 days after challenge. All dead fish are removed from the tank and the livers are removed and plated to enumerate the presence of the challenge organism. Efficacy is measured as a degree of livability comparing vaccinates to non-vaccinated controls.

Example 9

Characterization of Metal Regulated Proteins of Y. enterocolitica ATCC Strain 27729 and Y. pestis Strain KIM6+

The proteins of the composition prepared as described in example 1 from Y. enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ were characterized using matrix assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF MS). Samples of each composition were was resolved using a 10% sodium dodecyl sulfate-polyacrylamide gel. After the proteins of a composition had been resolved, the gel was stained with coomasie brilliant blue to visualize the proteins.

Materials and Methods

Excision and Washing.

The gel was washed for 10 minutes with water twice. Each protein band of interest was excised by cutting as close to the protein band as possible to reduce the amount of gel present in the sample. Each gel slice was cut into 1.times.1 mm cubes and placed in 1.5 ml tube. The gel pieces were washed with water for 15 minutes. All the solvent volumes used in the wash steps were approximately equal to twice the volume of the gel slice. The gel slice was next washed with water/acetonitrile (1:1) for 15 minutes. The water/acetonitrile mixture was removed, and acetonitrile was added to cover until the gel pieces turned a sticky white, at which time the acetonitrile was removed. The gel pieces were rehydrated in 100 mM NH.sub.4HCO.sub.3, and after 5 minutes, a volume of acetonitrile equal to twice the volume of the gel pieces was added. This was incubated for 15 minutes, the liquid removed, and the gel pieces dried in a SpeedVac.

Reduction & Alkylation.

The dried gel pieces were rehydrated in 10 mM DTT and 100 mM NH.sub.4HCO.sub.3, and incubated for 45 minutes at 56.degree. C. After allowing the tubes to cool to room temperature, the liquid was removed and the same volume of a mixture of 55 mM iodoacetamide and 100 mM NH.sub.4HCO.sub.3 was immediately added. This was incubated for 30 minutes at room temperature in the dark. The liquid was removed, acetonitrile was added to cover until the gel pieces turned a sticky white, at which time the acetonitrile was removed. The gel pieces were rehydrated in 100 mM NH.sub.4HCO.sub.3, and after 5 minutes, a volume of acetonitrile equal to twice the volume of the gel pieces was added. This was incubated for 15 minutes, the liquid removed, and the gel pieces dried in a Speed vac. If residual coomassie still remained, the wash with 100 mM NH.sub.4HCO.sub.3/acetonitrile was repeated.

In-Gel Digestion.

Gel pieces were completely dried down in a Speed Vac. The pieces were rehydrated in digestion buffer (50 mM NH.sub.4HCO.sub.3, 5 mM CaCl.sub.2, 12.5 nanograms per microliter (ng/.mu.l) trypsin) at 4.degree. C. Enough buffer was added to cover the gel pieces, and more was added as needed. The gel pieces were incubated on ice for 45 minutes, and the supernatant removed and replaced with 5-2 .mu.l of same buffer without trypsin. This was incubated at 37.degree. C. overnight in an air incubator.

Extraction of Peptides.

A sufficient volume of 25 mM NH.sub.4HCO.sub.3 was added to cover gel pieces, and incubated for 15 minutes (typically in a bath sonicator). The same volume of acetonitrile was added and incubated for 15 minutes (in a bath sonicator if possible), and the supernatant was recovered. The extraction was repeated twice, using 5% formic acid instead of NH.sub.4HCO.sub.3. A sufficient volume of 5% formic acid was added to cover gel pieces, and incubated for 15 minutes (typically in a bath sonicator). The same volume of acetonitrile was added and incubated for 15 minutes (typically in a bath sonicator), and the supernatant was recovered. The extracts were pooled, and 10 mM DTT was added to a final concentration of 1 mM DTT. The sample was dried in a SpeedVac to a final volume of approximately 5 .mu.l.

Desalting of Peptides.

The samples were desalted using ZIPTIP pipette tips (C18, Millipore, Billerica, Mass.) as suggested by the manufacturer. Briefly, a sample was reconstituted in reconstitution solution (5:95 acetonitrile:H.sub.2O, 0.1%-0.5% trifluoroacetic acid), centrifuged, and the pH checked to verify that it was less than 3. A ZIPTIP was hydrated by aspirating 10 .mu.l of solution 1 (50:50 acetonitrile:H.sub.2O, 0.1% trifluoroacetic acid) and discarding the aspirated aliquots. This was followed by aspirating 10 .mu.l of solution 2 (0.1% trifluoroacetic acid in deionized H.sub.2O) and discarding the aspirated aliquots. The sample was loaded into the tip by aspirating 10 .mu.l of the sample slowly into the tip, expelling it into the sample tube, and repeating this 5 to 6 times. Ten microliters of solution 2 was aspirated into the tip, the solution discarded by expelling, and this process was repeated 5-7 times to wash. The peptides were eluted by aspirating 2.5 .mu.l of ice cold solution 3 (60:40, acetonitrile:H.sub.2O, 0.1% trofluoroacetic acid), expelling, and then re-aspirating the same aliquot in and out of the tip 3 times. After the solution has been expelled from the tip, the tube is capped and stored on ice.

Mass Spectrometric Peptide Mapping.

The peptides were suspended in 10 .mu.l to 30 .mu.l of 5% formic acid, and analyzed by MALDI-TOF MS (Bruker Daltonics Inc., Billerica, Mass.). The mass spectrum of the peptide fragments was determined as suggested by the manufacturer. Briefly, a sample containing the peptides resulting from a tryptic digest were mixed with matrix cyano-4-hydroxycinnamic acid, transferred to a target, and allowed to dry. The dried sample was placed in the mass spectrometer, irradiated, and the time of flight of each ion detected and used to determine a peptide mass fingerprint for each protein present in the composition. Known polypeptides (human angiotensin II, monoisotopic mass MH.sup.+1046.5 (Sigma Chemical Co.), and adenocorticotropin hormone fragment 18-39, MH.sup.+2465.2 (Sigma Chemical Co.)) were used to standardize the machine.

Data Analysis.

The experimentally observed masses for the peptides in each mass spectrum were compared to the expected masses of resulting from known proteins using the Peptide Mass Fingerprint search method of the Mascot search engine (Matrix Science Ltd., London, UK, and www.matrixscience.com, see Perkins et al., Electrophoresis 20, 3551-3567 (1999)). The search parameters included: database, NCBInr; taxonomy, bacteria (eubacteria); type of search, peptide mass fingerprint; enzyme, trypsin; fixed modifications, none; variable modifications, none or oxidized methionine; mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance, .+-.1 Da or .+-.1 330 ppm; peptide charge state, Mr; max missed cleavages, 1; number of queries, 25.

Results

The result of this search was a mass fingerprint for protein present in the composition (Tables 5 and 6).

TABLE-US-00005 TABLE 5 Experimental data from MALDI-TOF MS analysis of proteins isolated from Y. enterocolitica ATCC strain 27729. Approximate molecular m/z value of polypeptide Polypeptide weight in kilodaltons fragments resulting from Designation (kDa).sup.1 trypsin digestion.sup.2 Lw545 268 929.46 1140.47 1312.57 1440.69 1526.68 1555.66 1581.70 1596.67 1683.69 2110.21 Lw391A (.+-.1 Da) 83 687.5 976.4 1001.6 1016.5 1141.6 1170.7 1171.7 1198.5 1344.5 1357.7 1395.6 1453.7 1477.7 1521.7 1693.8 1716.8 1829.8 1962.0 2014.1 2020.0 2042.0 2164.1 2226.1 2417.3 3175.5 Lw391B (.+-.1 Da) 83 1001.6 1104.6 1140.6 1155.5 1171.7 1209.5 1214.7 1338.6 1453.7 1568.8 1634.9 1651.8 1660.9 1709.8 1750.0 1851.0 1988.1 2105.1 2112.1 2164.1 2387.2 2453.1 2538.4 3423.7 Lw392 (.+-.1 Da 79 837.5 1018.6 1071.5 1086.5 1132.7 1189.5 1215.6 1236.6 1256.6 1264.6 1361.6 1497.7 1502.8 1615.7 1653.8 1718.9 1770.9 1820.9 1828.1 2006.0 2067.1 2120.9 2300.3 2308.2 2783.3 2912.4 3024.5 3287.6 Lw393 (.+-.1 Da) 70 714.6 760.5 807.5 820.5 920.5 1024.6 1052.6 1187.6 1200.6 1395.7 1437.7 1480.7 1541.9 1546.9 1565.8 1668.8 1732.0 1790.9 1906.0 1982.2 1984.1 1997.1 2011.1 2028.2 2060.2 2134.1 2163.3 2275.4 2364.3 2378.5 2384.3 2564.4 2658.4 2834.7 2930.7 Lw550 66 868.6500 882.5700 884.5900 1021.7000 1087.7100 1168.7300 1177.8200 1208.6800 1346.8700 1750.0100 1755.0500 1852.2800 2521.8100 2607.6700 2944.1000 3087.0800 Lw552 45 1140.5500 1209.5400 1312.5800 1440.6400 1501.6900 1526.6200 1581.6800 1596.6800 Lw555 37 705.3700 881.2400 971.1700 1122.3100 1280.1900 1295.2200 1335.2900 1510.3000 1908.5300 2245.7300 2324.7100 2642.7500 2985.0200 3087.9700 Lw557 31 864.49 1404.50 1616.68 1780.68 1876.82 2071.04 2379.08 .sup.1Molecular weight, in kilodaltons, of polypeptide obtained from Y. enterocolitica ATCC strain 27729. .sup.2m/z, mass (m) to charge (z) ratio.

TABLE-US-00006 TABLE 6 Experimental data from MALDI-TOF MS analysis of proteins isolated from Y. pestis strain KIM6+. Approximate molecular m/z value of polypeptide Polypeptide weight in kilodaltons fragments resulting from Designation (kDa).sup.1 trypsin digestion.sup.2 Lw529 104 644.50 685.40 771.40 841.40 899.50 962.40 1137.40 1277.40 1293.40 1386.40 1410.50 1422.60 1498.60 1567.50 1679.70 1684.60 1726.70 1873.70 1991.70 2020.80 2182.80 2584.90 2843.20 Lw530 99 1191.40 1514.50 1591.50 1597.50 1637.50 1671.50 1714.60 1719.60 1751.60 1820.60 1863.70 1967.60 2122.60 Lw531 94 962.20 1168.20 1258.30 1372.30 1384.30 1409.30 1521.40 1669.50 1686.40 1714.40 1717.40 1797.50 1833.50 1845.50 2218.60 2426.60 Lw532 88 889.30 927.30 946.40 961.40 1172.40 1177.40 1290.40 1333.50 1358.40 1404.50 1419.50 1508.50 1579.60 1673.60 1736.60 2401.00 2666.00 Lw533 77 687.40 785.40 859.30 953.40 1141.50 1156.50 1171.50 1198.40 1403.50 1409.50 1483.50 1523.50 1551.60 1618.60 1675.50 1746.60 1788.70 1820.70 1852.80 1941.60 2013.90 2018.80 2057.80 2168.80 2170.00 2427.00 2457.80 2829.10 Lw534 73 629.40 749.40 910.30 931.40 1292.50 1371.50 1441.40 1479.50 1587.60 1605.60 1641.60 1655.50 1706.60 1708.60 1758.70 1797.80 1856.80 1913.70 2004.80 2072.80 2155.90 2301.90 2395.90 2484.90 2558.20 2676.20 2984.10 3162.30 3185.30 3425.50 3472.40 Lw535 64 714.40 760.40 774.40 807.40 920.40 1024.40 1052.40 1103.40 1165.40 1187.40 1200.40 1282.50 1395.40 1445.50 1480.50 1546.60 1668.50 1693.60 1731.60 1790.60 1905.70 1969.70 1981.80 2010.80 2027.80 2059.80 2163.00 2363.90 2378.10 2820.20 2930.20 Lw536 60 1011.46 1187.55 1231.54 1238.57 1291.57 1567.76 1605.78 1621.74 1669.68 2021.02 2397.21 Lw537 46 873.53 1001.53 1180.50 1258.60 1300.67 1307.58 1325.59 1368.72 1395.70 1436.67 1609.91 1616.82 1780.94 1952.05 1959.02 2020.04 2099.15 2178.22 2710.51 Lw538 44 776.51 837.65 905.62 1027.71 1073.74 1200.79 1232.67 1233.72 1290.81 1376.71 1603.90 1615.01 1711.08 1774.04 1796.13 1906.14 1978.16 2001.23 Lw683 37 691.26 894.21 911.21 1050.26 1115.19 1120.19 1122.24 1198.17 1263.19 1308.24 1320.34 1423.28 1437.31 1491.23 1534.41 1579.39 2245.71 2367.68 2487.63 2684.79 2980.02 3292.91 Lw541 31 1020.84 1075.77 1203.86 1248.88 1322.87 1404.95 1788.29 1991.60 2092.61 2119.74 Lw542 31 1143.91 1299.97 1309.09

1341.97 1372.04 1580.12 1781.45 1791.43 1954.57 2191.78 2632.11 Lw544 20 807.40 1114.43 1210.48 1244.46 1259.51 1270.49 1357.49 1790.90 2003.91 2989.45 .sup.1Molecular weight, in kilodaltons, of polypeptide obtained from Y. pestis strain KIM6+. .sup.2m/z, mass (m) to charge (z) ratio.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

SEQUENCE LISTINGS

1

4511422PRTYersinia enterocolitica 1Met Thr Lys Asp Phe Lys Ile Ser Val Ser Ala Ala Leu Ile Ser Ala 1 5 10 15 Leu Phe Ser Ser Pro Tyr Ala Phe Ala Asn Asn Asp Glu Val His Phe 20 25 30 Thr Ala Val Gln Ile Ser Pro Asn Ser Asp Pro Asp Ser His Val Met 35 40 45 Ile Phe Gln Pro Glu Val Arg Ala Pro Gly Gly Thr Asn Ala Leu Ala 50 55 60 Lys Gly Thr His Ser Ile Ala Val Gly Ala Ser Ala Glu Ala Ala Glu 65 70 75 80 Arg Ala Ala Val Ala Val Gly Ala Gly Ser Ile Ala Thr Gly Val Asn 85 90 95 Ser Val Ala Ile Gly Pro Leu Ser Lys Ala Leu Gly Asp Ser Ala Val 100 105 110 Thr Tyr Gly Ala Gly Ser Thr Ala Gln Lys Asp Gly Val Ala Ile Gly 115 120 125 Ala Arg Ala Ser Thr Ser Asp Thr Gly Val Ala Val Gly Phe Asn Ser 130 135 140 Lys Val Asp Ala Lys Asn Ser Val Ser Ile Gly His Ser Ser His Val 145 150 155 160 Ala Val Asp His Asp Tyr Ser Ile Ala Ile Gly Asp Arg Ser Lys Thr 165 170 175 Asp Arg Lys Asn Ser Val Ser Ile Gly His Glu Ser Leu Asn Arg Gln 180 185 190 Leu Thr His Leu Ala Ala Gly Thr Lys Asp Thr Asp Ala Val Asn Val 195 200 205 Ala Gln Leu Lys Lys Glu Ile Glu Lys Thr Gln Glu Asn Ala Asn Lys 210 215 220 Lys Ser Ala Glu Val Leu Gly Ile Ala Asn Asn Tyr Thr Asp Ser Lys 225 230 235 240 Ser Ala Glu Thr Leu Glu Asn Ala Arg Lys Glu Ala Phe Asp Leu Ser 245 250 255 Asn Asp Ala Leu Asp Met Ala Lys Lys His Ser Asn Ser Val Ala Arg 260 265 270 Thr Thr Leu Glu Thr Ala Glu Glu His Thr Asn Lys Lys Ser Ala Glu 275 280 285 Thr Leu Ala Ser Ala Asn Val Tyr Ala Asp Ser Lys Ser Ser His Thr 290 295 300 Leu Lys Thr Ala Asn Ser Tyr Thr Asp Val Thr Val Ser Asn Ser Thr 305 310 315 320 Lys Lys Ala Ile Arg Glu Ser Asn Gln Tyr Thr Asp His Lys Phe His 325 330 335 Gln Leu Asp Asn Arg Leu Asp Lys Leu Asp Thr Arg Val Asp Lys Gly 340 345 350 Leu Ala Ser Ser Ala Ala Leu Asn Ser Leu Phe Gln Pro Tyr Gly Val 355 360 365 Gly Lys Val Asn Phe Thr Ala Gly Val Gly Gly Tyr Arg Ser Ser Gln 370 375 380 Ala Leu Ala Ile Gly Ser Gly Tyr Arg Val Asn Glu Ser Val Ala Leu 385 390 395 400 Lys Ala Gly Val Ala Tyr Ala Gly Ser Ser Asp Val Met Tyr Asn Ala 405 410 415 Ser Phe Asn Ile Glu Trp 420 2686PRTYersinia enterocolitica 2Met Pro Arg Ser Thr Ser Asp Arg Phe Arg Trp Ser Pro Leu Ser Leu 1 5 10 15 Ala Ile Ala Cys Thr Leu Ser Leu Ala Val Gln Ala Ala Asp Thr Ser 20 25 30 Ser Thr Gln Thr Asn Ser Lys Lys Arg Ile Ala Asp Thr Met Val Val 35 40 45 Thr Ala Thr Gly Asn Glu Arg Ser Ser Phe Glu Ala Pro Met Met Val 50 55 60 Thr Val Val Glu Ala Asp Thr Pro Thr Ser Glu Thr Ala Thr Ser Ala 65 70 75 80 Thr Asp Met Leu Arg Asn Ile Pro Gly Leu Thr Val Thr Gly Ser Gly 85 90 95 Arg Val Asn Gly Gln Asp Val Thr Leu Arg Gly Tyr Gly Lys Gln Gly 100 105 110 Val Leu Thr Leu Val Asp Gly Ile Arg Gln Gly Thr Asp Thr Gly His 115 120 125 Leu Asn Ser Thr Phe Leu Asp Pro Ala Leu Val Lys Arg Val Glu Ile 130 135 140 Val Arg Gly Pro Ser Ala Leu Leu Tyr Gly Ser Gly Ala Leu Gly Gly 145 150 155 160 Val Ile Ser Tyr Glu Thr Val Asp Ala Ala Asp Leu Leu Leu Pro Gly 165 170 175 Gln Asn Ser Gly Tyr Arg Val Tyr Ser Ala Ala Ala Thr Gly Asp His 180 185 190 Ser Phe Gly Leu Gly Ala Ser Ala Phe Gly Arg Thr Asp Asp Val Asp 195 200 205 Gly Ile Leu Ser Phe Gly Thr Arg Asp Ile Gly Asn Ile Arg Gln Ser 210 215 220 Asp Gly Phe Asn Ala Pro Asn Asp Glu Thr Ile Ser Asn Val Leu Ala 225 230 235 240 Lys Gly Thr Trp Arg Ile Asp Gln Ile Gln Ser Leu Ser Ala Asn Leu 245 250 255 Arg Tyr Tyr Asn Asn Ser Ala Leu Glu Pro Lys Asn Pro Gln Thr Ser 260 265 270 Ala Ala Ser Ser Thr Asn Leu Met Thr Asp Arg Ser Thr Ile Gln Arg 275 280 285 Asp Ala Gln Leu Lys Tyr Asn Ile Lys Pro Leu Asp Gln Glu Trp Leu 290 295 300 Asn Ala Thr Ala Gln Val Tyr Tyr Ser Glu Val Glu Ile Asn Ala Arg 305 310 315 320 Pro Gln Gly Thr Pro Glu Glu Gly Arg Lys Gln Thr Thr Lys Gly Gly 325 330 335 Lys Leu Glu Asn Arg Thr Arg Leu Phe Thr Asp Ser Phe Ala Ser His 340 345 350 Leu Leu Thr Tyr Gly Thr Glu Ala Tyr Lys Gln Glu Gln Thr Pro Ser 355 360 365 Gly Ala Thr Glu Ser Phe Pro Gln Ala Asp Ile Arg Phe Gly Ser Gly 370 375 380 Trp Leu Gln Asp Glu Ile Thr Leu Arg Asp Leu Pro Val Ser Ile Leu 385 390 395 400 Ala Gly Thr Arg Tyr Asp Asn Tyr Arg Gly Ser Ser Glu Gly Tyr Ala 405 410 415 Asp Val Asp Ala Asp Lys Trp Ser Ser Arg Gly Ala Val Ser Val Thr 420 425 430 Pro Thr Asp Trp Leu Met Leu Phe Gly Ser Tyr Ala Gln Ala Phe Arg 435 440 445 Ala Pro Thr Met Gly Glu Met Tyr Asn Asp Ser Lys His Phe Ser Met 450 455 460 Asn Ile Trp Val Thr Pro Asp Gln Leu Leu Gly Thr Asn Pro Asn Leu 465 470 475 480 Lys Pro Glu Thr Asn Glu Thr Gln Glu Tyr Gly Phe Gly Leu Arg Phe 485 490 495 Asn Asp Leu Met Met Ala Glu Asp Asp Leu Gln Phe Lys Ala Ser Tyr 500 505 510 Phe Asp Thr Asn Ala Lys Asp Tyr Ile Ser Thr Gly Val Thr Met Asp 515 520 525 Phe Gly Phe Gly Pro Gly Gly Leu Tyr Cys Lys Asn Cys Ser Thr Tyr 530 535 540 Ser Thr Asn Ile Asp Arg Ala Lys Ile Trp Gly Trp Asp Ala Thr Met 545 550 555 560 Thr Tyr Gln Thr Gln Trp Phe Asn Leu Gly Leu Ala Tyr Asn Arg Thr 565 570 575 Arg Gly Lys Asn Gln Asn Thr Asn Glu Trp Leu Asp Thr Ile Asn Pro 580 585 590 Asp Thr Val Thr Ser Thr Leu Asp Val Pro Val Ala Asn Ser Gly Phe 595 600 605 Ala Val Gly Trp Ile Gly Thr Phe Ala Asp Arg Ser Ser Arg Val Ser 610 615 620 Ser Ser Gly Thr Pro Gln Ala Gly Tyr Gly Val Asn Asp Phe Tyr Val 625 630 635 640 Ser Tyr Lys Gly Gln Glu Gln Phe Lys Gly Met Thr Thr Thr Val Val 645 650 655 Leu Gly Asn Ala Phe Asp Lys Gly Tyr Tyr Gly Pro Gln Gly Val Pro 660 665 670 Gln Asp Gly Arg Asn Ala Lys Phe Phe Val Ser Tyr Gln Trp 675 680 685 3758PRTYersinia enterocolitica 3Met Asn Gln Thr Ile Ser Ser Arg Ala Pro Gln Lys Arg Leu Ala Pro 1 5 10 15 Arg Leu Leu Cys Val Met Ile Gly Ala Ala Leu Gly Thr Leu Ser Ala 20 25 30 Ser Ser Trp Ala Ala Ala Ala Thr Asp Ser Thr Ala Glu Asn Ala Lys 35 40 45 Lys Thr Ser Ala Thr Ala Ala Thr Ala Lys Ala Glu Asp Ser Lys Thr 50 55 60 Asn Asp Thr Ile Thr Val Val Gly Ala Gln Glu Thr Phe Arg Ala Gly 65 70 75 80 Gly Asn Asp Leu Ile Pro Thr Tyr Leu Asp Gly Gln Val Ala Asn Gly 85 90 95 Gly Arg Ile Gly Phe Leu Gly Gln Gln Asp Ala Arg Asn Val Pro Phe 100 105 110 Asn Val Ile Gly Tyr Thr Ser Lys Met Ile Glu Asp Gln Gln Ala Asn 115 120 125 Ser Ile Ala Asp Val Val Lys Asn Asp Ala Ser Val Gln Asn Val Arg 130 135 140 Gly Tyr Gly Asn Pro Ser Gln Asn Tyr Arg Ile Arg Gly Tyr Asn Leu 145 150 155 160 Asp Gly Asp Asp Ile Ser Phe Gly Gly Leu Phe Gly Val Leu Pro Arg 165 170 175 Gln Ile Val Ser Thr Ser Met Val Glu Arg Val Glu Val Phe Lys Gly 180 185 190 Ala Asn Ala Phe Ile Asn Gly Ile Ser Pro Ser Gly Ser Gly Val Gly 195 200 205 Gly Met Ile Asn Leu Glu Pro Lys Arg Ala Gly Asp Thr Pro Leu Thr 210 215 220 Arg Val Thr Val Asp Tyr Gly Ser Ala Ser Gln Val Gly Gly Ala Leu 225 230 235 240 Asp Val Gly Arg Arg Tyr Gly Asp Asp Asp Gln Phe Gly Val Arg Val 245 250 255 Asn Val Leu His Arg Glu Gly Glu Ser Ala Ile His Asp Gln Lys Glu 260 265 270 Arg Thr Thr Ala Val Ser Thr Gly Leu Asp Tyr Arg Gly Asp Arg Ala 275 280 285 Arg Thr Ser Leu Asp Val Gly Tyr Gln Lys Gln Thr Ile His His Met 290 295 300 Arg Thr Asp Val Ala Ile Gly Gly Ala Thr Val Ile Pro Glu Pro Pro 305 310 315 320 Ser Ser Thr Leu Asn Tyr Gly Gln Ser Trp Val Tyr Thr Asp Met Glu 325 330 335 Thr Thr Phe Gly Met Leu Arg Ser Glu Tyr Asp Val Ser Gln Asn Trp 340 345 350 Thr Val Tyr Gly Ser Val Gly Ala Ser Arg Asn Glu Glu Thr Gly Gln 355 360 365 Tyr Gly Ala Pro Met Leu Thr Asn Asn Asn Gly Asp Ala Thr Ile Ser 370 375 380 Arg Leu Tyr Val Pro Tyr Val Ala Asp Ser Val Ala Gly Leu Gly Gly 385 390 395 400 Ile Arg Gly His Phe Asp Thr Gly Pro Ile Thr His Lys Val Asn Leu 405 410 415 Gly Tyr Ala Ala Asn Tyr Arg Thr Thr Lys Ser Ala Trp Asn Met Ser 420 425 430 Gly Gln Glu Asp Thr Asn Ile Tyr Asn Pro Gly Val Ile Gly Phe Pro 435 440 445 Gln Thr Val Met Gly Ser Asp Ser Gln Asp Pro Gln Leu Thr Ser Gln 450 455 460 Val Arg Ala Ser Gly Leu Ser Leu Ser Asp Thr Leu Ser Met Met Asp 465 470 475 480 Asp Lys Val Ser Leu Met Leu Gly Val Arg Arg Gln Glu Val Thr Ile 485 490 495 Arg Asn Phe Asp Ser Gly Val Pro Asn Ser Ala Gly Ser Leu Asp Ala 500 505 510 Met Lys Val Thr Pro Ile Tyr Gly Ile Met Val Lys Pro Trp Glu Lys 515 520 525 Val Ser Leu Tyr Ala Asn His Ile Glu Ala Leu Gly Pro Gly Lys Ser 530 535 540 Ala Pro Tyr Gln Tyr Asn Gly Lys Pro Val Val Asn Ala Gly Gln Ile 545 550 555 560 Pro Gly Ile Ile His Ser Lys Gln Asn Glu Ile Gly Val Lys Phe Asp 565 570 575 Asn Gln Arg Tyr Gly Gly Thr Leu Ala Leu Phe Glu Ile Thr Arg Pro 580 585 590 Thr Gly Met Val Asp Pro Ala Thr Asn Val Tyr Gly Phe Tyr Gly Glu 595 600 605 Gln Arg Asn Arg Gly Ile Glu Leu Asn Val Phe Gly Glu Pro Val Phe 610 615 620 Gly Thr Arg Leu Leu Ala Ser Ala Thr Trp Leu Asp Pro Lys Leu Thr 625 630 635 640 Lys Ala Ala Asp Ser Ala Asn Asn Gly Asn Asp Ala Val Gly Val Ala 645 650 655 Asn Tyr Gln Leu Val Phe Gly Gly Glu Tyr Asp Ile Pro Val Val Glu 660 665 670 Gly Leu Thr Ala Thr Gly Thr Val Val Arg Ser Gly Ser Gln Tyr Ala 675 680 685 Asn Glu Ala Asn Thr Leu Lys Leu Lys Pro Trp Thr Arg Leu Asp Leu 690 695 700 Gly Val Arg Tyr Thr Met Pro Met Lys Asp Thr Ser Leu Thr Trp Arg 705 710 715 720 Ala Asn Ile Glu Asn Val Thr Asn Glu Arg Tyr Trp Glu Ser Val Glu 725 730 735 Asp Ser Gly Thr Tyr Ile Tyr Gln Gly Asp Pro Arg Ala Leu Lys Leu 740 745 750 Ser Val Ser Met Asp Phe 755 4710PRTYersinia enterocolitica 4Met Phe Ser Ala Phe Ile Ile Lys Arg Ser Ala Ile Leu Cys Ser Leu 1 5 10 15 Ala Met Phe Ile Pro Leu Ala Ser Ile Ala Asp Asp Thr Ile Glu Val 20 25 30 Thr Ala Lys Ala Gly His Glu Ala Asp Leu Pro Thr Ser Gly Tyr Thr 35 40 45 Ala Thr Thr Thr Lys Gly Ala Thr Lys Thr Asp Gln Pro Leu Ile Leu 50 55 60 Thr Ala Gln Ser Val Ser Val Val Thr Arg Gln Gln Met Asp Asp Gln 65 70 75 80 Asn Val Ala Thr Val Asn Gln Ala Leu Asn Tyr Thr Pro Gly Val Phe 85 90 95 Thr Gly Phe Ser Gly Gly Ala Thr Arg Tyr Asp Thr Val Ala Leu Arg 100 105 110 Gly Phe His Gly Gly Asp Val Asn Asn Thr Phe Leu Asp Gly Leu Arg 115 120 125 Leu Leu Ser Asp Gly Gly Ser Tyr Asn Val Leu Gln Val Asp Pro Trp 130 135 140 Phe Leu Glu Arg Ile Asp Val Ile Lys Gly Pro Ser Ser Ala Leu Tyr 145 150 155 160 Gly Gln Ser Ile Pro Gly Gly Val Val Met Met Thr Ser Lys Arg Pro 165 170 175 Gln Phe Thr Ser Glu Gly His Phe Arg Leu Thr Ala Gly Asn Asn Asn 180 185 190 Thr Gln Val Ala Ala Phe Asp Tyr Thr Asp Ala Ile Ser Glu His Trp 195 200 205 Ala Phe Arg Leu Thr Gly Ile Thr Arg Asn Ser Asp Thr Met Tyr Asp 210 215 220 His Gln Arg Glu Glu Arg Tyr Ala Ile Ala Pro Ser Leu Leu Trp Gln 225 230 235 240 Pro Asp Glu Asn Thr Ser Leu Leu Leu Arg Ala Asn Leu Gln Lys Asp 245 250 255 Pro Ser Gly Gly Tyr His Ser Ala Val Pro Ala Asp Gly Ser Ile Tyr 260 265 270 Gly Gln Lys Leu Ser Arg Gly Phe Phe Asp Gly Glu Ser Asn His Asn 275 280 285 Val Phe Lys Arg Trp Gln Gln Ile Tyr Ser Tyr Glu Phe Ser His Lys 290 295 300 Phe Asp Asp Val Trp Ser Phe Arg Gln Asn Ala Ser Tyr Thr His Ser 305 310 315 320 Asn Thr Gln Leu Glu Gln Val Tyr Gln Gly Gly Trp Asn Ser Asp Arg 325 330 335 Thr Leu Met Asn Arg Tyr Tyr Ser Gly Glu Asp Ser Ser Leu Asn Ala 340 345 350 Phe Ala Val Asp Asn Gln Leu Glu Ala Asp Leu Arg Thr Ala Ala Val 355 360 365 Lys His Lys Val Leu Leu Gly Val Asp Phe Gln Lys Phe Arg Asn Asn 370 375 380 Leu Arg Ser Asp Ser Ala Tyr Ala Thr Pro Leu Asn Pro Tyr Thr Gly 385 390 395 400 Val Ser Gly Gly Ser Thr Leu Tyr Ser Asp Tyr Leu Leu Thr Thr Pro 405 410 415 Gly Ile Asn Thr Ser Tyr Leu Ser Arg Arg Tyr Glu Gln Ser Gly

Val 420 425 430 Tyr Leu Gln Asp Glu Met Thr Leu Asp Asn Trp His Leu Asn Leu Ser 435 440 445 Gly Arg Tyr Asp Arg Met Lys Thr Glu Asn Ile Asn Asn Thr Ala Asn 450 455 460 Ser Thr Asp Glu Arg Thr Asp Asn His Ala Ser Gly Arg Ala Ser Leu 465 470 475 480 Leu Tyr Ser Phe Asp Ser Gly Ile Ser Pro Tyr Val Ser Tyr Ser Gln 485 490 495 Ala Ile Thr Pro Ser Leu Phe Pro Asp Ala Gln Gln Lys Leu Leu Lys 500 505 510 Pro Met Thr Ser Glu Gln Tyr Glu Val Gly Ile Ile Tyr Gln Pro Pro 515 520 525 Gly Ser Thr Ser Leu Tyr Ser Ala Ala Leu Tyr Asp Leu Thr Gln Asn 530 535 540 Asp Val Ala Asn Arg Ala Val Pro Ala Thr Tyr Tyr Val Pro Ala Gly 545 550 555 560 Lys Val Asn Ser Gln Gly Leu Glu Leu Glu Ala Arg Ser Gln Ile Ser 565 570 575 Asp Arg Leu Ser Val Ile Ala Gly Tyr Thr Tyr Asn Arg Val Lys Phe 580 585 590 Lys Asp Ala Ile Asp Gly Asn Asp Gly Asn Thr Pro Val Leu Ala Pro 595 600 605 Ser Asn Met Ala Ser Leu Trp Ala Gln Tyr Glu Ala Gly Tyr Gly Ile 610 615 620 Asn Val Gly Ala Gly Ile Arg Tyr Ile Gly Lys Gln Trp Ala Asp Asp 625 630 635 640 Ala Asn Thr Leu Arg Val Pro Ser Tyr Thr Leu Gly Asp Ala Ser Val 645 650 655 Arg Ala Asp Leu Gly Thr Trp Ala Ala Ser Leu Lys Gly Ala Phe Val 660 665 670 Gln Leu Asn Val Asn Asn Ile Ala Asp Lys Lys Tyr Val Ala Ala Cys 675 680 685 Tyr Ser Thr Ser Tyr Cys Tyr Trp Gly Ala Glu Arg Ser Val Gln Ala 690 695 700 Thr Val Gly Tyr Asp Phe 705 710 5673PRTYersinia enterocolitica 5Met Lys Met Thr Arg Leu Tyr Pro Leu Ala Leu Gly Gly Leu Leu Leu 1 5 10 15 Pro Ala Ile Ala Asn Ala Gln Thr Ser Gln Gln Asp Glu Ser Thr Leu 20 25 30 Glu Val Thr Ala Ser Lys Gln Ser Ser Arg Ser Ala Ser Ala Asn Asn 35 40 45 Val Ser Ser Thr Val Val Ser Ala Pro Glu Leu Ser Asp Ala Gly Val 50 55 60 Thr Ala Ser Asp Lys Leu Pro Arg Val Leu Pro Gly Leu Asn Ile Glu 65 70 75 80 Asn Ser Gly Asn Met Leu Phe Ser Thr Ile Ser Leu Arg Gly Val Ser 85 90 95 Ser Ala Gln Asp Phe Tyr Asn Pro Ala Val Thr Leu Tyr Val Asp Gly 100 105 110 Val Pro Gln Leu Ser Thr Asn Thr Ile Gln Ala Leu Thr Asp Val Gln 115 120 125 Ser Val Glu Leu Leu Arg Gly Pro Gln Gly Thr Leu Tyr Gly Lys Ser 130 135 140 Ala Gln Gly Gly Ile Ile Asn Ile Val Thr Gln Gln Pro Asp Ser Thr 145 150 155 160 Pro Arg Gly Tyr Ile Glu Gly Gly Val Ser Ser Arg Asp Ser Tyr Arg 165 170 175 Ser Lys Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly 180 185 190 Ser Val Thr Leu Leu Arg Gln Val Asp Asp Gly Asp Met Ile Asn Pro 195 200 205 Ala Thr Gly Ser Asp Asp Leu Gly Gly Thr Arg Ala Ser Ile Gly Asn 210 215 220 Val Lys Leu Arg Leu Ala Pro Asp Asp Gln Pro Trp Glu Met Gly Phe 225 230 235 240 Ala Ala Ser Arg Glu Cys Thr Arg Ala Thr Gln Asp Ala Tyr Val Gly 245 250 255 Trp Asn Asp Ile Lys Gly Arg Lys Leu Ser Leu Ser Asp Gly Ser Pro 260 265 270 Asp Pro Tyr Met Arg Arg Cys Thr Asp Ser Gln Thr Leu Ser Gly Lys 275 280 285 Tyr Thr Thr Asp Asp Trp Val Phe Asn Leu Ile Ser Ala Trp Gln Gln 290 295 300 Gln His Tyr Ser Arg Thr Phe Pro Ser Gly Ser Leu Ile Val Asn Met 305 310 315 320 Pro Gln Arg Trp Asn Gln Asp Val Gln Glu Leu Arg Ala Ala Thr Leu 325 330 335 Gly Asp Ala Arg Thr Val Asp Met Val Phe Gly Leu Tyr Arg Gln Asn 340 345 350 Thr Arg Glu Lys Leu Asn Ser Ala Tyr Asp Met Pro Thr Met Pro Tyr 355 360 365 Leu Ser Ser Thr Gly Tyr Thr Thr Ala Glu Thr Leu Ala Ala Tyr Ser 370 375 380 Asp Leu Thr Trp His Leu Thr Asp Arg Phe Asp Ile Gly Gly Gly Val 385 390 395 400 Arg Phe Ser His Asp Lys Ser Ser Thr Gln Tyr His Gly Ser Met Leu 405 410 415 Gly Asn Pro Phe Gly Asp Gln Gly Lys Ser Asn Asp Asp Gln Val Leu 420 425 430 Gly Gln Leu Ser Ala Gly Tyr Met Leu Thr Asp Asp Trp Arg Val Tyr 435 440 445 Thr Arg Ile Ala Gln Gly Tyr Lys Pro Ser Gly Tyr Asn Ile Val Pro 450 455 460 Thr Ala Gly Leu Asp Ala Lys Pro Phe Val Ala Glu Lys Ser Ile Asn 465 470 475 480 Tyr Glu Leu Gly Thr Arg Tyr Glu Thr Ala Asp Val Thr Leu Gln Ala 485 490 495 Ala Thr Phe Tyr Thr His Thr Lys Asp Met Gln Leu Tyr Ser Gly Pro 500 505 510 Val Gly Met Gln Thr Leu Ser Asn Ala Gly Lys Ala Asp Ala Thr Gly 515 520 525 Val Glu Leu Glu Ala Lys Trp Arg Phe Ala Pro Gly Trp Ser Trp Asp 530 535 540 Ile Asn Gly Asn Val Ile Arg Ser Glu Phe Thr Asn Asp Ser Glu Leu 545 550 555 560 Tyr His Gly Asn Arg Val Pro Phe Val Pro Arg Tyr Gly Ala Gly Ser 565 570 575 Ser Val Asn Gly Val Ile Asp Thr Arg Tyr Gly Ala Leu Met Pro Arg 580 585 590 Leu Ala Val Asn Leu Val Gly Pro His Tyr Phe Asp Gly Asp Asn Gln 595 600 605 Leu Arg Gln Gly Thr Tyr Ala Thr Leu Asp Ser Ser Leu Gly Trp Gln 610 615 620 Ala Thr Glu Arg Ile Asn Ile Ser Val His Val Asp Asn Leu Phe Asp 625 630 635 640 Arg Arg Tyr Arg Thr Tyr Gly Tyr Met Asn Gly Ser Ser Ala Val Ala 645 650 655 Gln Val Asn Met Gly Arg Thr Val Gly Ile Asn Thr Arg Ile Asp Phe 660 665 670 Phe 6623PRTYersinia bercovieri 6Met Val Thr Ala Ser Gly Phe Gln Gln Arg Ile Gln Asp Ser Ala Ala 1 5 10 15 Ser Ile Ser Val Val Thr Arg Glu Gln Ile Glu Asn Lys Ala Tyr Thr 20 25 30 Asp Ile Thr Asp Ala Leu Lys Asp Val Pro Gly Val Val Val Thr Gly 35 40 45 Gly Gly Ser His Ser Asp Ile Ser Ile Arg Gly Met Ala Ala Lys Tyr 50 55 60 Thr Leu Ile Leu Val Asp Gly Lys Arg Val Asp Thr Arg Gly Thr Arg 65 70 75 80 Pro Asn Ser Asp Gly Ser Gly Ile Glu Gln Gly Trp Leu Pro Pro Leu 85 90 95 Ala Ala Ile Glu Arg Ile Glu Val Val Arg Gly Pro Met Ser Ser Leu 100 105 110 Tyr Gly Ser Asp Ala Met Gly Gly Val Ile Asn Val Ile Thr Arg Lys 115 120 125 Val Gly Lys Glu Trp His Gly Thr Val Arg Ala Asp Ala Thr Leu Gln 130 135 140 Glu Asp Ser Lys Ser Gly Asp Ile Phe Gln Thr Asn Ala Tyr Ala Ser 145 150 155 160 Gly Pro Leu Ile Asp Gly Leu Leu Gly Leu Lys Val Ser Gly Leu Leu 165 170 175 Ser His Arg Ser Glu Asp Lys Ile Val Asp Gly Tyr Asn Glu Gln Arg 180 185 190 Leu Arg Asn Gly Ala Ala Thr Phe Thr Leu Thr Pro Asp Asp Lys Asn 195 200 205 Glu Phe Asp Phe Asp Ile Gly His Tyr Val Gln Asp Arg Asn Ser Thr 210 215 220 Ala Gly Arg Ser Val Ala Leu Asn Gly Lys Ser Ser Asp Val Gln Tyr 225 230 235 240 Asp Arg Asn Asn Tyr Ala Ile Thr His His Gly Tyr Tyr Asp Phe Gly 245 250 255 Asn Ser Thr Ser Tyr Val Gln Arg Asp Glu Thr Arg Asn Pro Ser Arg 260 265 270 Glu Met Lys Ser Val Asp Asn Ile Phe Asn Thr Gln Thr Ser Phe Leu 275 280 285 Leu Asp Asn His Thr Leu Ile Leu Gly Gly Gln Tyr Arg Tyr Glu Glu 290 295 300 Leu Asn Asp Thr Gly Asn Gln Leu Ala Ser Ala Lys Asp Leu Thr Lys 305 310 315 320 Leu Thr Arg Trp Ser Trp Ala Leu Phe Ala Glu Asp Glu Trp Gln Met 325 330 335 Thr Asn Asp Phe Ala Leu Thr Gly Gly Val Arg Met Asp Gln Asp Glu 340 345 350 Asn Tyr Gly Thr His Trp Thr Pro Arg Leu Tyr Gly Val Trp His Leu 355 360 365 Ala Glu Gln Trp Thr Leu Lys Gly Gly Val Ser Gly Gly Tyr Arg Ser 370 375 380 Pro Asp Leu Arg Gln Ala Thr Glu Asn Trp Gly Gln Ile Thr Gly Gly 385 390 395 400 Arg Gly Asp Pro Ala Ile Ile Ile Gly Asn Ala Asn Leu Lys Pro Glu 405 410 415 Arg Ser Ile Ser Gln Glu Ile Gly Ile Leu Trp Asp Asp Gln Glu Gly 420 425 430 Met Asn Ala Gly Val Thr Leu Phe Asn Thr Asp Phe Lys Asp Lys Ile 435 440 445 Thr Glu Val Arg Arg Cys Thr Asp Thr Thr Gly Lys Ala Ser Gly Gln 450 455 460 Cys Met Ile Asn Gly Ala Ser Tyr Lys Phe Ile Ser Asp Arg Thr Asn 465 470 475 480 Val Asp Lys Ala Ile Thr Arg Gly Val Glu Ala Thr Phe Gly Trp Asp 485 490 495 Ile Asn Gln Glu Trp Ser Leu Thr Ser Asn Tyr Thr Phe Thr Gln Ser 500 505 510 Glu Gln Lys Ser Gly Gln Phe Ala Gly Gln Pro Leu Asn Gln Met Pro 515 520 525 Lys His Met Leu Asn Gly Thr Leu Asn Trp Gln Ala Ser Glu Ala Leu 530 535 540 Ala Thr Trp Val Arg Ala Asn Tyr Arg Gly Lys Thr Ser Glu Tyr Leu 545 550 555 560 Asn Arg Thr Ser Ile Gly Gly Ser Thr Pro Ser Tyr Thr Phe Val Asp 565 570 575 Leu Gly Ala Asn Tyr Gln Leu Thr Lys Glu Phe Arg Leu Met Gly Gly 580 585 590 Val Tyr Asn Val Leu Asp Lys Arg Val Asp Ile Glu Val Asn Asp Lys 595 600 605 Val Leu Asp Gly Arg Arg Tyr Met Val Gly Ala Ser Tyr Asp Phe 610 615 620 7422PRTYersinia enterocolitica 7Met Thr Lys Asp Phe Lys Ile Ser Val Ser Ala Ala Leu Ile Ser Ala 1 5 10 15 Leu Phe Ser Ser Pro Tyr Ala Phe Ala Asn Asn Asp Glu Val His Phe 20 25 30 Thr Ala Val Gln Ile Ser Pro Asn Ser Asp Pro Asp Ser His Val Met 35 40 45 Ile Phe Gln Pro Glu Val Arg Ala Pro Gly Gly Thr Asn Ala Leu Ala 50 55 60 Lys Gly Thr His Ser Ile Ala Val Gly Ala Ser Ala Glu Ala Ala Glu 65 70 75 80 Arg Ala Ala Val Ala Val Gly Ala Gly Ser Ile Ala Thr Gly Val Asn 85 90 95 Ser Val Ala Ile Gly Pro Leu Ser Lys Ala Leu Gly Asp Ser Ala Val 100 105 110 Thr Tyr Gly Ala Gly Ser Thr Ala Gln Lys Asp Gly Val Ala Ile Gly 115 120 125 Ala Arg Ala Ser Thr Ser Asp Thr Gly Val Ala Val Gly Phe Asn Ser 130 135 140 Lys Val Asp Ala Lys Asn Ser Val Ser Ile Gly His Ser Ser His Val 145 150 155 160 Ala Val Asp His Asp Tyr Ser Ile Ala Ile Gly Asp Arg Ser Lys Thr 165 170 175 Asp Arg Lys Asn Ser Val Ser Ile Gly His Glu Ser Leu Asn Arg Gln 180 185 190 Leu Thr His Leu Ala Ala Gly Thr Lys Asp Thr Asp Ala Val Asn Val 195 200 205 Ala Gln Leu Lys Lys Glu Ile Glu Lys Thr Gln Glu Asn Ala Asn Lys 210 215 220 Lys Ser Ala Glu Val Leu Gly Ile Ala Asn Asn Tyr Thr Asp Ser Lys 225 230 235 240 Ser Ala Glu Thr Leu Glu Asn Ala Arg Lys Glu Ala Phe Asp Leu Ser 245 250 255 Asn Asp Ala Leu Asp Met Ala Lys Lys His Ser Asn Ser Val Ala Arg 260 265 270 Thr Thr Leu Glu Thr Ala Glu Glu His Thr Asn Lys Lys Ser Ala Glu 275 280 285 Thr Leu Ala Ser Ala Asn Val Tyr Ala Asp Ser Lys Ser Ser His Thr 290 295 300 Leu Lys Thr Ala Asn Ser Tyr Thr Asp Val Thr Val Ser Asn Ser Thr 305 310 315 320 Lys Lys Ala Ile Arg Glu Ser Asn Gln Tyr Thr Asp His Lys Phe His 325 330 335 Gln Leu Asp Asn Arg Leu Asp Lys Leu Asp Thr Arg Val Asp Lys Gly 340 345 350 Leu Ala Ser Ser Ala Ala Leu Asn Ser Leu Phe Gln Pro Tyr Gly Val 355 360 365 Gly Lys Val Asn Phe Thr Ala Gly Val Gly Gly Tyr Arg Ser Ser Gln 370 375 380 Ala Leu Ala Ile Gly Ser Gly Tyr Arg Val Asn Glu Ser Val Ala Leu 385 390 395 400 Lys Ala Gly Val Ala Tyr Ala Gly Ser Ser Asp Val Met Tyr Asn Ala 405 410 415 Ser Phe Asn Ile Glu Trp 420 8366PRTYersinia bercovieri 8Met Lys Leu Arg Val Leu Ser Leu Leu Val Pro Ala Leu Leu Val Ala 1 5 10 15 Gly Ser Ala Gly Ala Ala Glu Ile Tyr His Lys Asp Gly Asn Lys Leu 20 25 30 Asp Leu Tyr Gly Lys Val Asp Gly Leu His Tyr Phe Ser Asp Asp Lys 35 40 45 Ser Lys Asp Gly Asp Gln Ser Tyr Met Arg Phe Gly Leu Lys Gly Glu 50 55 60 Thr Gln Ile Ser Asp Gln Leu Thr Gly Tyr Gly Gln Trp Glu Tyr Gln 65 70 75 80 Ala Asn Leu Asn Lys Ala Glu Asp Gln Asp Gln Gly Asn Phe Thr Arg 85 90 95 Leu Gly Phe Ala Gly Leu Lys Phe Ala Asp Tyr Gly Ser Leu Asp Tyr 100 105 110 Gly Arg Asn Tyr Gly Val Leu Tyr Asp Val Thr Ser Trp Thr Asp Val 115 120 125 Leu Pro Glu Phe Gly Gly Asp Thr Tyr Gly Ala Asp Asn Phe Met Ser 130 135 140 Gln Arg Ala Asn Gly Leu Ala Thr Tyr Arg Asn Thr Asn Phe Phe Gly 145 150 155 160 Leu Val Asp Gly Leu Asn Phe Ala Leu Gln Tyr Gln Gly Lys Asn Gly 165 170 175 Ser Pro Thr Glu Ser Asn Asn Gly Arg Asp Val Lys Gly Gln Asn Gly 180 185 190 Asp Gly Tyr Gly Met Ser Leu Ser Tyr Asp Leu Gly Trp Gly Val Ser 195 200 205 Ala Ala Ala Ala Met Ser Ser Ser Lys Arg Thr Thr Glu Gln Asn Gln 210 215 220 Leu Leu Phe Gly Asn Gly Asp Arg Ala Asp Ala Tyr Ser Gly Gly Leu 225 230 235 240 Lys Tyr Asp Ala Asn Asn Val Tyr Leu Ala Ala Thr Tyr Ala Gln Thr 245 250 255 Tyr Asn Leu Thr Arg Phe Gly Asn Phe Gln Asn Asn Asn Ser Gly Phe 260 265 270 Ala Asn Lys Ala Gln Asn Ile Glu Leu Val Ala Gln Tyr Gln Phe Asp 275 280

285 Phe Gly Leu Arg Pro Ser Val Ala Tyr Leu Gln Ser Lys Gly Lys Asp 290 295 300 Leu Gly Asn Gly Tyr Gly Asp Gln Asp Leu Val Gln Tyr Val Asp Val 305 310 315 320 Gly Ala Thr Tyr Phe Phe Asn Lys Asn Met Ser Thr Tyr Val Asp Tyr 325 330 335 Lys Ile Asn Leu Leu Asp Glu Asn Glu Phe Thr Lys Asn Ala Gly Ile 340 345 350 Asn Thr Asp Asp Ile Val Ala Val Gly Leu Val Tyr Gln Phe 355 360 365 9245PRTYersinia enterocolitica 9Met Lys Lys Asn Met Lys Leu Ile Ala Ile Thr Ala Val Leu Ser Ser 1 5 10 15 Val Leu Val Leu Ser Gly Cys Gly Ala Met Ser Thr Ala Ile Lys Lys 20 25 30 Arg Asn Leu Glu Val Lys Thr Gln Met Ser Glu Thr Ile Trp Leu Glu 35 40 45 Pro Ser Ser Gln Lys Thr Val Tyr Leu Gln Ile Lys Asn Thr Ser Asp 50 55 60 Lys Asn Met Leu Gly Leu Ala Pro Lys Ile Thr Lys Ala Val Gln Asp 65 70 75 80 Lys Gly Tyr Thr Val Thr Ser Ser Pro Glu Asp Ala His Tyr Trp Ile 85 90 95 Gln Ala Asn Val Leu Lys Ala Asp Lys Met Asp Leu Arg Glu Ala Glu 100 105 110 Gly Phe Leu Ser Gln Gly Tyr Gln Gly Ala Ala Leu Gly Ala Ala Leu 115 120 125 Gly Ala Gly Ile Thr Gly Tyr Asn Ser Asn Ser Ala Gly Ala Ser Leu 130 135 140 Gly Val Gly Leu Ala Ala Gly Leu Val Gly Met Val Ala Asp Ala Met 145 150 155 160 Val Glu Asp Ile Asn Tyr Thr Met Val Thr Asp Val Gln Ile Ser Glu 165 170 175 Lys Thr Asp Thr Pro Leu Gln Thr Asp Asn Val Ala Ala Leu Lys Gln 180 185 190 Gly Thr Ser Gly Tyr Lys Val Gln Thr Ser Thr Gln Thr Gly Asn Lys 195 200 205 His Gln Tyr Gln Thr Arg Val Val Ser Ser Ala Asn Lys Val Asn Leu 210 215 220 Lys Phe Glu Glu Ala Gln Pro Val Leu Glu Asp Gln Leu Ala Lys Ser 225 230 235 240 Ile Ala Asn Ile Leu 245 10891PRTYersinia pestis 10Met Ala Val Thr Asn Val Ala Glu Leu Asn Glu Leu Val Ala Arg Val 1 5 10 15 Lys Lys Ala Gln Arg Glu Tyr Ala Asn Phe Ser Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40 45 Leu Ala Lys Leu Ala Val Thr Glu Ser Gly Met Gly Ile Val Glu Asp 50 55 60 Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr 65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Ile Leu Cys Glu Asp Lys Thr Phe Gly 85 90 95 Thr Ile Thr Ile Ala Glu Pro Ile Gly Leu Ile Cys Gly Ile Val Pro 100 105 110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ala Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Gly Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp 130 135 140 Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala 145 150 155 160 Gly Ala Pro Ala Asp Ile Ile Gly Trp Ile Asp Ala Pro Thr Val Glu 165 170 175 Leu Ser Asn Gln Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala 180 185 190 Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro 195 200 205 Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Val Asp Glu Thr 210 215 220 Ala Asp Ile Lys Arg Val Val Ala Ser Ile Leu Met Ser Lys Thr Phe 225 230 235 240 Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Ile Val Val Asp 245 250 255 Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Ser His Gly Gly Tyr 260 265 270 Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Ile Ile Leu Lys 275 280 285 Asn Gly Gly Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Thr Lys Ile 290 295 300 Ala Glu Met Ala Gly Ile Lys Val Pro Ser Asn Thr Lys Ile Leu Ile 305 310 315 320 Gly Glu Val Lys Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys 325 330 335 Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asn Phe Glu Glu Ala 340 345 350 Val Glu Lys Ala Glu Lys Leu Val Glu Met Gly Gly Ile Gly His Thr 355 360 365 Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Thr Ala Arg Val Lys Tyr 370 375 380 Phe Gly Asp Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala 385 390 395 400 Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser 405 410 415 Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn 420 425 430 Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala 435 440 445 Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg 450 455 460 Gly Ser Leu Pro Ile Ala Leu Glu Glu Val Ala Thr Asp Gly Ala Lys 465 470 475 480 Arg Ala Phe Ile Val Thr Asp Arg Tyr Leu Phe Asn Asn Gly Tyr Ala 485 490 495 Asp Gln Val Thr Ser Val Leu Lys Ser His Gly Ile Glu Thr Glu Val 500 505 510 Phe Phe Glu Val Glu Ala Ala Pro Thr Leu Ser Ile Val Arg Lys Gly 515 520 525 Ala Glu Gln Met Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly 530 535 540 Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu 545 550 555 560 His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile 565 570 575 Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Leu 580 585 590 Val Ala Ile Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe 595 600 605 Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp 610 615 620 Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met 625 630 635 640 Asn Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr 645 650 655 His Ala Leu Glu Ala Tyr Val Ser Val Leu Ala Asn Glu Tyr Ser Asp 660 665 670 Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Phe Leu Pro Ala 675 680 685 Ser Tyr Asn Glu Gly Ala Lys Asn Pro Val Ala Arg Glu Arg Val His 690 695 700 Asn Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly 705 710 715 720 Val Cys His Ser Met Ala His Lys Leu Gly Ser Glu Phe His Ile Pro 725 730 735 His Gly Leu Ala Asn Ala Met Leu Ile Ser Asn Val Ile Arg Tyr Asn 740 745 750 Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg 755 760 765 Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu 770 775 780 Ser Ala Pro Gly Asp Arg Thr Ala Gln Lys Ile Gln Lys Leu Leu Ala 785 790 795 800 Trp Leu Asp Glu Ile Lys Ala Glu Leu Gly Ile Pro Ala Ser Ile Arg 805 810 815 Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Lys Val Asp Lys Leu 820 825 830 Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr 835 840 845 Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Met Asp Thr Tyr Tyr Gly 850 855 860 Arg Glu Tyr Val Glu Glu Phe Asp Arg Glu Glu Glu Val Ala Ala Ala 865 870 875 880 Thr Ala Pro Lys Ala Glu Lys Lys Thr Lys Lys 885 890 11702PRTYersinia pseudotuberculosis 11Met Ala Arg Lys Thr Pro Ile Glu Arg Tyr Arg Asn Ile Gly Ile Ser 1 5 10 15 Ala His Ile Asp Ala Gly Lys Thr Thr Thr Thr Glu Arg Ile Leu Phe 20 25 30 Tyr Thr Gly Val Asn His Lys Ile Gly Glu Val His Asp Gly Ala Ala 35 40 45 Thr Met Asp Trp Met Glu Gln Glu Gln Glu Arg Gly Ile Thr Ile Thr 50 55 60 Ser Ala Ala Thr Thr Cys Phe Trp Ser Gly Met Ala Lys Gln Phe Glu 65 70 75 80 Pro His His Val Asn Ile Ile Asp Thr Pro Gly His Val Asp Phe Thr 85 90 95 Ile Glu Val Glu Arg Ser Met Arg Val Leu Asp Gly Ala Val Met Val 100 105 110 Tyr Cys Ala Val Gly Gly Val Gln Pro Gln Ser Glu Thr Val Trp Arg 115 120 125 Gln Ala Asn Lys Tyr Lys Val Pro Arg Ile Ala Phe Val Asn Lys Met 130 135 140 Asp Arg Met Gly Ala Asn Phe Leu Arg Val Val Gly Gln Leu Lys Ser 145 150 155 160 Arg Leu Gly Ala Asn Pro Val Pro Leu Gln Leu Ala Ile Gly Ala Glu 165 170 175 Glu Lys Phe Thr Gly Ile Ile Asp Leu Val Lys Met Lys Ala Ile Asn 180 185 190 Trp Asn Glu Ala Asp Gln Gly Val Thr Phe Glu Tyr Glu Glu Ile Pro 195 200 205 Ala Asp Met Ala Glu Leu Ala Ala Glu Trp His Gln Asn Leu Val Glu 210 215 220 Ser Ala Ala Glu Ala Ser Asp Glu Leu Met Asp Lys Tyr Leu Gly Gly 225 230 235 240 Glu Glu Leu Thr Glu Glu Glu Ile Lys Lys Ala Leu Arg Gln Arg Val 245 250 255 Leu Lys Ser Glu Ile Ile Leu Val Thr Cys Gly Ser Ala Phe Lys Asn 260 265 270 Lys Gly Val Gln Ala Met Leu Asp Ala Val Ile Glu Tyr Leu Pro Ala 275 280 285 Pro Thr Asp Val Glu Ser Ile Asn Gly Ile Leu Asp Asp Gly Lys Asp 290 295 300 Thr Pro Ala Val Arg His Ser Asp Asp Lys Glu Pro Phe Ser Ala Leu 305 310 315 320 Ala Phe Lys Ile Ala Thr Asp Pro Phe Val Gly Asn Leu Thr Phe Phe 325 330 335 Arg Val Tyr Ser Gly Ile Val Asn Ser Gly Asp Thr Val Leu Asn Ser 340 345 350 Val Lys Ser Gln Arg Glu Arg Leu Gly Arg Ile Val Gln Met His Ala 355 360 365 Asn Lys Arg Glu Glu Ile Lys Glu Val His Ala Gly Asp Ile Ala Ala 370 375 380 Ala Ile Gly Leu Lys Asp Val Thr Thr Gly Asp Thr Leu Cys Asp Pro 385 390 395 400 Asn Asn Pro Ile Ile Leu Glu Arg Met Glu Phe Pro Glu Pro Val Ile 405 410 415 Ser Val Ala Val Glu Pro Lys Thr Lys Ala Asp Gln Glu Lys Met Gly 420 425 430 Met Ala Leu Gly Arg Leu Ala Lys Glu Asp Pro Ser Phe Arg Val Trp 435 440 445 Thr Asp Glu Glu Ser Gly Gln Thr Ile Ile Ala Gly Met Gly Glu Leu 450 455 460 His Leu Asp Ile Leu Val Asp Arg Met Arg Arg Glu Phe Asn Val Glu 465 470 475 480 Ala Asn Val Gly Lys Pro Gln Val Ala Tyr Arg Glu Thr Ile Arg Glu 485 490 495 Thr Val Lys Asp Val Glu Gly Lys His Ala Lys Gln Ser Gly Gly Arg 500 505 510 Gly Gln Tyr Gly His Val Val Ile Asp Met Ser Pro Leu Pro Pro Gly 515 520 525 Gly Val Gly Tyr Glu Phe Val Asn Glu Ile Val Gly Gly Ser Ile Pro 530 535 540 Lys Glu Phe Ile Pro Ala Val Asp Lys Gly Ile Gln Glu Gln Leu Lys 545 550 555 560 Ser Gly Pro Leu Ala Gly Tyr Pro Val Val Asp Val Lys Val Arg Leu 565 570 575 His Tyr Gly Ser Tyr His Asp Val Asp Ser Ser Glu Leu Ala Phe Lys 580 585 590 Leu Ala Gly Ser Ile Ala Phe Lys Glu Gly Phe Lys Arg Ala Lys Pro 595 600 605 Val Leu Leu Glu Pro Ile Met Lys Val Glu Val Glu Thr Pro Glu Asp 610 615 620 Tyr Met Gly Asp Val Met Gly Asp Leu Asn Arg Arg Arg Gly Ile Ile 625 630 635 640 Glu Gly Met Glu Asp Thr Ala Thr Gly Lys Thr Val Arg Val Lys Val 645 650 655 Pro Leu Ser Glu Met Phe Gly Tyr Ala Thr Asp Leu Arg Ser Gln Thr 660 665 670 Gln Gly Arg Ala Ser Tyr Ser Met Glu Phe Leu Glu Tyr Ala Glu Ala 675 680 685 Pro Ser Asn Val Ala Lys Ala Val Ile Glu Ala Arg Gly Lys 690 695 700 12815PRTYersinia pestis 12Met Thr Ser Pro Phe Ser Tyr Thr Ser Pro Val Val Ser Val Asp Ala 1 5 10 15 Leu Lys His Ser Ile Ala Tyr Lys Leu Met Phe Ile Ile Gly Lys Asp 20 25 30 Pro Thr Ile Ala Thr Gln His Asp Trp Leu Asn Ala Thr Leu Phe Ala 35 40 45 Val Arg Asp Arg Met Val Glu Arg Trp Leu Arg Ser Asn Arg Ala Gln 50 55 60 Leu Ser Gln Asp Val Arg Gln Val Tyr Tyr Leu Ser Met Glu Phe Leu 65 70 75 80 Leu Gly Arg Thr Leu Ser Asn Ala Leu Leu Ser Met Gly Ile Tyr Asp 85 90 95 Glu Ile Glu Gln Ala Leu Asp Glu Met Gly Leu Ser Leu Ser Glu Leu 100 105 110 Leu Lys Glu Glu Asn Asp Pro Gly Leu Gly Asn Gly Gly Leu Gly Arg 115 120 125 Leu Ala Ala Cys Phe Leu Asp Ser Leu Ala Thr Leu Ala Leu Pro Gly 130 135 140 Arg Gly Tyr Gly Ile Arg Tyr Glu Tyr Gly Met Phe Ser Gln Lys Ile 145 150 155 160 Val Asn Gly Gln Gln Met Glu Ser Pro Asp Asn Trp Leu Glu Tyr Gly 165 170 175 Asn Ala Trp Glu Phe Pro Arg His Asn Thr Arg Tyr Lys Val Arg Phe 180 185 190 Gly Gly Arg Ile Gln Gln Glu Gly Ser Lys Ile Arg Trp Leu Glu Thr 195 200 205 Glu Glu Ile Leu Ala Cys Ala Tyr Asp Gln Ile Ile Pro Gly Phe Asp 210 215 220 Thr Asp Ala Thr Asn Thr Leu Arg Leu Trp Ser Ala Gln Ala Ser Asn 225 230 235 240 Glu Ile Asn Leu Gly Lys Phe Asn Gln Gly Asp Tyr Phe Ala Ala Val 245 250 255 Glu Asp Lys Asn His Ser Glu Asn Val Ser Arg Val Leu Tyr Pro Asp 260 265 270 Asp Ser Thr Tyr Ser Gly Arg Glu Leu Arg Leu Arg Gln Glu Tyr Phe 275 280 285 Leu Val Ser Ala Thr Val Gln Asp Ile Leu Asn Arg His Trp Ala Met 290 295 300 His His Thr Phe Asn Asn Leu Ala Asp Lys Ile Ala Ile His Leu Asn 305 310 315 320 Asp Thr His Pro Val Leu Ser Ile Pro Glu Met Met Arg Leu Leu Ile 325 330 335 Asp Glu His Lys Phe Thr Trp Met Asp Ala Trp Asp Val Val Gln Gln 340 345 350 Val Phe Ser Tyr Thr Asn His Thr Leu Met Ser Glu Ala Leu Glu Thr 355 360 365 Trp Pro Val Asp Met Ile Gly

Lys Ile Leu Pro Arg His Leu Gln Ile 370 375 380 Ile Phe Asp Ile Asn Asp His Phe Leu Lys Leu Val Glu Glu Gln Tyr 385 390 395 400 Pro Asp Asp Lys Glu Leu Leu Ser Arg Val Ser Val Ile Asp Glu Asn 405 410 415 Asn Gly Arg Arg Ile Arg Met Ala Trp Leu Ala Val Ile Ala Ser His 420 425 430 Lys Val Asn Gly Val Ser Ala Leu His Ser Glu Leu Met Val Gln Ser 435 440 445 Leu Phe Ala Asp Phe Ala Arg Ile Phe Pro Asn Arg Phe Cys Asn Lys 450 455 460 Thr Asn Gly Val Thr Pro Arg Arg Trp Leu Gly Leu Ala Asn Arg Pro 465 470 475 480 Leu Ala Ala Val Leu Asp Asp Ser Ile Gly Gln Thr Trp Arg Thr Asp 485 490 495 Leu Ser Gln Leu Ser Glu Leu Glu Lys Asn Leu Asp Tyr Pro Ser Phe 500 505 510 Leu Leu Ala Leu Gln Lys Ala Lys Leu Glu Asn Lys Lys Arg Leu Ala 515 520 525 Val Tyr Ile Ala Glu Lys Leu Asn Ile Val Val Asn Pro Ala Ala Leu 530 535 540 Phe Asp Val Gln Ile Lys Arg Ile His Glu Tyr Lys Arg Gln Leu Leu 545 550 555 560 Asn Val Leu His Val Ile Thr Arg Tyr Asn Arg Ile Ile Asp Ala Pro 565 570 575 Asp Asn Asn Trp Val Pro Arg Val Val Ile Phe Ala Gly Lys Ala Ala 580 585 590 Ser Ala Tyr Tyr Asn Ala Lys Gln Ile Ile His Leu Ile Asn Asp Val 595 600 605 Ala Lys Val Ile Asn Asn Asp Pro Arg Ile Asn Asn Leu Leu Lys Val 610 615 620 Val Phe Ile Pro Asn Tyr Ser Val Ser Leu Ala Gln Leu Ile Ile Pro 625 630 635 640 Ala Ala Asp Leu Ser Glu Gln Ile Ser Leu Ala Gly Thr Glu Ala Ser 645 650 655 Gly Thr Ser Asn Met Lys Phe Ala Leu Asn Gly Ala Leu Thr Ile Gly 660 665 670 Thr Leu Asp Gly Ala Asn Val Glu Ile Arg Glu His Val Gly Glu Glu 675 680 685 Asn Ile Phe Ile Phe Gly Asn Thr Thr Glu Gln Val Glu Ala Leu Arg 690 695 700 Lys Ser Gly Tyr Asn Pro Arg Lys Tyr Tyr Asp Glu Asp Pro Glu Leu 705 710 715 720 His Gln Val Leu Thr Gln Ile Ala Thr Gly Thr Phe Ser Pro Glu Glu 725 730 735 Pro His Arg Tyr Thr Asn Leu Phe Asp Ser Leu Val Asn Leu Gly Asp 740 745 750 His Tyr Gln Leu Leu Ala Asp Tyr Arg Ser Tyr Val Asp Thr Gln Glu 755 760 765 Gln Val Asp Ala Leu Tyr Arg Asn Arg Asp Glu Trp Ser Arg Lys Thr 770 775 780 Leu Leu Asn Ile Ala Asn Met Gly Tyr Phe Ser Ser Asp Arg Thr Ile 785 790 795 800 Lys Glu Tyr Ala Asp Glu Ile Trp His Ile Lys Pro Ile Arg Leu 805 810 815 13780PRTYersinia pestis 13Met Lys Lys Arg Phe Pro Thr Leu Leu Ala Thr Leu Ile Trp Thr Ala 1 5 10 15 Leu Tyr Ser Gln His Thr Leu Ala Asp Leu Ala Glu Gln Cys Met Leu 20 25 30 Gly Val Pro Thr Tyr Asp Gln Pro Leu Val Thr Gly Asp Pro Asn Gln 35 40 45 Leu Pro Val Arg Ile Asn Ala Asp Lys Thr Glu Ala Asn Tyr Pro Asp 50 55 60 Asn Ala Leu Phe Thr Gly Asn Val Ile Val Gln Gln Gly Asn Ser Thr 65 70 75 80 Leu Thr Ala Asn Gln Val Glu Leu Thr Gln Val Gln Lys Pro Gly Glu 85 90 95 Val Ile Pro Leu Arg Thr Val Thr Ala Thr Gly Asp Val Asn Tyr Asp 100 105 110 Asp Pro Gln Ile Lys Leu Lys Gly Pro Lys Gly Trp Ser Asn Leu Asn 115 120 125 Thr Lys Asp Thr Asp Met Asp Lys Gly Lys Tyr Gln Met Val Gly Arg 130 135 140 Gln Gly Arg Gly Asp Ala Asp Leu Met Lys Leu Arg Asp Gln Ser Arg 145 150 155 160 Tyr Thr Ile Leu Lys Asn Gly Thr Phe Thr Ser Cys Leu Pro Gly Asp 165 170 175 Asn Ser Trp Ser Val Val Gly Ser Glu Val Ile His Asp Arg Glu Glu 180 185 190 Gln Val Val Glu Val Trp Asn Ala Arg Phe Lys Ile Gly Lys Val Pro 195 200 205 Val Phe Tyr Ser Pro Tyr Met Gln Leu Pro Val Gly Asp Lys Arg Arg 210 215 220 Ser Gly Phe Leu Ile Pro Asn Ala Lys Phe Thr Ser Asn Asn Gly Phe 225 230 235 240 Glu Phe Leu Leu Pro Tyr Tyr Trp Asn Ile Ala Pro Asn Phe Asp Ala 245 250 255 Thr Ile Thr Pro His Tyr Met Glu Arg Arg Gly Leu Gln Trp Gln Asn 260 265 270 Glu Phe Arg Tyr Leu Leu Ala Pro Gly Ser Gly Thr Met Ala Leu Asp 275 280 285 Trp Leu Pro Asn Asp Arg Ile Tyr Thr Gly Pro Asp Gly Thr Asp Lys 290 295 300 Asn Ala Thr Arg Trp Leu Tyr Tyr Trp Gly His Ser Gly Val Met Asp 305 310 315 320 Gln Val Trp Arg Phe Asn Ile Asn Tyr Thr Arg Val Ser Asp Pro Ala 325 330 335 Tyr Phe Thr Asp Leu Thr Ser Gln Tyr Gly Ser Thr Thr Asp Gly Tyr 340 345 350 Ala Thr Gln Ile Phe Thr Ala Gly Tyr Ala Asn Glu Asn Trp Asn Ala 355 360 365 Thr Leu Ser Ser Lys Gln Phe Gln Val Phe Thr Ala Ala Gly Asn Ser 370 375 380 Asn Ala Tyr Arg Ala Gln Pro Gln Leu Asp Met Asn Tyr Tyr Lys Asn 385 390 395 400 Asp Val Gly Pro Phe Asp Met His Val Tyr Gly Gln Ala Ala Lys Phe 405 410 415 Thr Ser Val Asn Pro Thr Asn Pro Glu Ala Ser Arg Phe His Ile Glu 420 425 430 Pro Thr Val Asn Leu Pro Leu Ser Asn Ser Trp Gly Ser Ile Asn Thr 435 440 445 Glu Ala Lys Leu Leu Ala Thr His Tyr Gln Gln Asp Ile Pro Ala Ser 450 455 460 Phe Ala Asp Asn Ala Ser Asn Pro Lys Leu Lys Asp Ser Val Asn Arg 465 470 475 480 Val Leu Pro Gln Phe Lys Val Asp Gly Lys Val Val Phe Asp Arg Ser 485 490 495 Met Asp Trp Ala Thr Gly Phe Thr Gln Thr Leu Glu Pro Arg Ala Gln 500 505 510 Tyr Leu Tyr Val Pro Tyr Arg Asn Gln Asp Asp Ile Tyr Ile Tyr Asp 515 520 525 Thr Thr Leu Met Gln Ser Asp Tyr Ser Gly Leu Phe Arg Asp Arg Thr 530 535 540 Tyr Ser Gly Leu Asp Arg Ile Ala Ser Ala Asn Gln Val Ser Thr Gly 545 550 555 560 Leu Thr Ser Arg Ile Tyr Asp Asp Ala Arg Val Glu Arg Phe Asn Val 565 570 575 Ser Val Gly Gln Ile Tyr Tyr Phe Ser Arg Ser Arg Thr Gly Asn Thr 580 585 590 Glu Ala Ile Asp Asn Ser Asn Ala Thr Gly Ser Leu Val Trp Ala Gly 595 600 605 Asp Thr Phe Trp Arg Ile Asn Asp Gln Leu Gly Leu Lys Gly Gly Ala 610 615 620 Gln Tyr Asp Thr Arg Leu Gly Ser Leu Thr Leu Gly Asn Ala Ile Met 625 630 635 640 Glu Tyr Arg Lys Asp Ala Asp Arg Met Ile Gln Leu Asn Tyr Arg Tyr 645 650 655 Ala Ser Pro Lys Tyr Ile Gln Ala Ala Val Pro Lys Val Tyr Asn Pro 660 665 670 Asp Tyr Gln Gln Gly Ile Ser Gln Val Gly Thr Thr Ala Ser Trp Pro 675 680 685 Ile Ala Asp Arg Trp Ala Ile Val Gly Ala Tyr Tyr Tyr Asp Thr Lys 690 695 700 Ala Lys Gln Pro Ala Ser Gln Leu Val Gly Leu Gln Tyr Asn Thr Cys 705 710 715 720 Cys Trp Ala Val Asn Leu Gly Tyr Glu Arg Lys Ile Thr Gly Trp Asn 725 730 735 Ala Gln Gly Gln Thr Ser Lys Tyr Asp Asn Lys Ile Gly Phe Asn Ile 740 745 750 Glu Leu Arg Gly Leu Ser Gly Gly His Ser Leu Gly Thr Ala Gln Met 755 760 765 Leu Asn Ser Gly Ile Leu Pro Tyr Gln Ser Ala Phe 770 775 780 14676PRTYersinia pestis 14Met Leu Arg Ser Thr Ser Asp Arg Phe Arg Trp Ser Ser Leu Ser Leu 1 5 10 15 Ala Ile Ala Cys Thr Leu Pro Leu Ala Thr Gln Ala Ala Asp Thr Thr 20 25 30 Thr Thr Gln Thr Ser Ser Lys Lys His Ser Thr Asp Thr Met Val Val 35 40 45 Thr Ala Thr Gly Asn Glu Arg Ser Ser Phe Glu Ala Pro Met Met Val 50 55 60 Thr Val Ile Glu Gly Asn Ala Pro Thr Ser Gln Thr Ala Ala Thr Ala 65 70 75 80 Ala Asp Met Leu Arg Gln Val Pro Gly Leu Thr Val Thr Gly Ser Gly 85 90 95 Arg Thr Asn Gly Gln Asp Val Val Met Arg Gly Tyr Gly Lys Gln Gly 100 105 110 Val Leu Thr Leu Val Asp Gly Val Arg Gln Gly Thr Asp Thr Gly His 115 120 125 Leu Asn Ser Thr Phe Leu Asp Pro Ala Leu Val Lys Arg Ile Glu Ile 130 135 140 Val Arg Gly Pro Ala Ala Leu Leu Tyr Gly Ser Gly Ala Leu Gly Gly 145 150 155 160 Val Ile Ala Tyr Glu Thr Val Asp Ala Ala Asp Met Leu Gln Pro Gly 165 170 175 Gln Asn Ser Gly Tyr Arg Val Tyr Ser Ser Ala Ala Thr Gly Asp His 180 185 190 Ser Phe Gly Leu Gly Ala Ser Ala Phe Gly Arg Thr Asp Asp Leu Asp 195 200 205 Gly Ile Leu Ser Phe Gly Thr Arg Asp Ile Gly Asn Ile Arg Gln Ser 210 215 220 Asn Gly Phe Asn Ala Pro Asn Asp Glu Thr Ile Ser Asn Val Leu Ala 225 230 235 240 Lys Gly Thr Trp Gln Ile Asp Ser Ile Gln Ser Leu Ser Ala Asn Leu 245 250 255 Arg Tyr Tyr Asn Asn Ser Ala Ile Glu Pro Lys Asn Pro Gln Thr Ser 260 265 270 Ala Pro Ser Ser Thr Asn Val Met Thr Asn Arg Ser Thr Ile Gln Arg 275 280 285 Asp Ala Gln Leu Arg Tyr Asn Ile Lys Pro Leu Asp Gln Glu Trp Leu 290 295 300 Asn Ala Thr Ala Gln Val Tyr Tyr Ser Glu Val Glu Ile Asn Ala Arg 305 310 315 320 Pro Gln Gly Ser Ala Glu Glu Gly Arg Glu Gln Thr Thr Glu Gly Val 325 330 335 Lys Leu Glu Asn Arg Thr Arg Leu Phe Ile Glu Ser Pro Ala Ser His 340 345 350 Leu Leu Thr Tyr Gly Thr Glu Thr Tyr Lys Gln Glu Gln Thr Pro Gly 355 360 365 Gly Ala Thr Glu Ser Phe Pro Gln Ala Lys Ile Arg Phe Ser Ser Gly 370 375 380 Trp Leu Gln Asp Glu Ile Thr Leu Arg Asp Leu Pro Val Ser Ile Leu 385 390 395 400 Ala Gly Thr Arg Tyr Asp Asn Tyr Ser Gly Ser Ser Asp Gly Tyr Ala 405 410 415 Asp Val Asp Ala Asp Lys Trp Ser Ser Arg Gly Ala Ile Ser Ile Thr 420 425 430 Pro Thr Asp Trp Leu Met Leu Phe Gly Ser Tyr Ala Gln Ala Phe Arg 435 440 445 Ala Pro Thr Met Gly Glu Met Tyr Asn Asp Ser Lys His Phe Ala Ile 450 455 460 Pro Ile Arg Pro Gly Leu Thr Leu Thr Asn Tyr Trp Val Pro Asn Pro 465 470 475 480 Asn Leu Lys Pro Glu Thr Asn Glu Thr Gln Glu Tyr Gly Phe Gly Leu 485 490 495 Arg Phe Ser Asp Leu Leu Met Ala Glu Asp Asp Leu Gln Phe Lys Val 500 505 510 Ser Tyr Phe Asp Thr Lys Ala Lys Asp Tyr Ile Ser Thr Arg Val Asp 515 520 525 Met Gln Ala Met Thr Thr Thr Ser Val Asn Ile Asp Gln Ala Lys Ile 530 535 540 Trp Gly Trp Asp Ala Ser Met Ser Tyr Lys Thr Ala Leu Phe Asn Trp 545 550 555 560 Asp Leu Ala Tyr Asn Arg Thr Arg Gly Lys Asn Gln Asn Thr Asp Glu 565 570 575 Trp Leu Asp Thr Ile Asn Pro Asp Thr Val Thr Ser Ile Val Asp Val 580 585 590 Pro Val Ala Asn Ser Gly Phe Ser Val Gly Trp Ile Gly Thr Phe Ala 595 600 605 Asn Arg Ser Ser Arg Val Ser Ser Ser Thr Pro Gln Ala Gly Tyr Gly 610 615 620 Val Asn Asp Phe Tyr Val Ser Tyr Lys Gly Gln Glu Ala Phe Lys Gly 625 630 635 640 Met Thr Thr Thr Met Leu Leu Gly Asn Val Phe Glu Lys Glu Tyr Tyr 645 650 655 Thr Pro Gln Gly Ile Pro Gln Asp Gly Arg Asn Val Lys Phe Phe Val 660 665 670 Ser Tyr Gln Trp 675 15698PRTYersinia pseudotuberculosis 15Met Ser Asn Lys Thr Ile Ala Phe Ala Leu Val Val Ala Ser Ser Ala 1 5 10 15 Pro Val Ile Ala Ala Asp Asn Asp Asn Ile Met Val Val Thr Ala Ser 20 25 30 Gly Tyr Glu Gln Lys Ile Arg Glu Ala Ala Ala Ser Ile Ser Val Ile 35 40 45 Ser Gln Asn Glu Leu Arg Gln Arg Asn Tyr Asn Asp Leu Ala Gln Ala 50 55 60 Leu Ser Asp Val Glu Gly Val Asp Val Asn Ser Ser Thr Gly Lys Thr 65 70 75 80 Gly Gly Leu Asp Ile Ser Ile Arg Gly Met Pro Ser Ala Tyr Thr Leu 85 90 95 Ile Leu Val Asp Gly Ile Arg Gln Asn Gly Thr Ser Asp Val Thr Pro 100 105 110 Asn Gly Phe Gly Ala Met Asn Thr Ser Phe Met Pro Pro Leu Ser Ala 115 120 125 Ile Glu Arg Ile Glu Val Ile Arg Gly Pro Met Ser Thr Leu Tyr Gly 130 135 140 Ser Asp Ala Ile Gly Gly Val Val Asn Ile Ile Thr Lys Lys Ile Thr 145 150 155 160 Lys Ala Trp Ala Ser Ser Ala Thr Leu Glu His Thr Phe Gln Glu Asn 165 170 175 Thr Ala Phe Gly Asp Ser Ser Lys Phe Ser Phe Tyr Ser Ser Gly Pro 180 185 190 Ala Val Glu Asp Gln Leu Gly Leu Ser Leu Arg Gly Thr Ile Phe Arg 195 200 205 Arg Asp Ala Ser Arg Val Glu Ser Ser Asn Thr Gly Val Glu Leu Ser 210 215 220 Arg Arg Gly Pro Asn Pro Val Lys Ala Asp Asn Tyr Asn Leu Gly Gly 225 230 235 240 Lys Leu Phe Trp Gln Leu Asn Thr Gln Ser Thr Leu Trp Leu Asp Gly 245 250 255 Asp Ile Ala Asn Gln Lys Tyr Asp Asn Ser Ala Asn Gln Leu Gly Thr 260 265 270 Ile Gly Ala Arg Gly Gly Tyr Glu Asp Thr Leu Arg Tyr Gln Arg Arg 275 280 285 Lys Ile Thr Leu Gly Asn Asp Asn Arg Leu Asp Phe Gly Thr Trp Asn 290 295 300 Ser Ser Leu Ser Tyr Asn Gln Thr Glu Asn Ile Gly Arg Leu Ile Thr 305 310 315 320 Asn Ala Ser Val Pro Gln Gly Ser Gly Leu Ala Gly Glu Lys Arg Leu 325 330 335 Leu Lys Asn Thr Asn Ile Ile Leu Asp Ser Lys Leu Val Ala Pro Leu 340 345 350 Gly Asp Asn His Met Val Thr Leu Gly Gly Gln Tyr Trp Asn Ala Ile 355 360 365 Met Lys Asp Gly Ile Val Leu Ala Asn Asn Gly Asp Glu Phe Ala Gln 370 375 380 Asp Ala Trp Ser Leu Phe Ser Glu Asp Glu Trp Arg Leu Leu Asp Ser 385 390

395 400 Leu Ala Leu Thr Tyr Gly Ala Arg Tyr Glu Tyr Gln Thr Thr Phe Gly 405 410 415 Gly His Ile Ser Pro Arg Ala Tyr Leu Val Trp Asp Ala Gln Asp Asn 420 425 430 Trp Thr Val Lys Gly Gly Val Ser Thr Gly Tyr Lys Thr Pro Thr Leu 435 440 445 Ala Gln Leu His Asn Gly Ile Ser Gly Val Thr Gly Gln Gly Thr Ile 450 455 460 Thr Thr Ile Gly Asn Pro Lys Leu Glu Pro Glu Ser Ser Val Asn Thr 465 470 475 480 Glu Val Gly Val Tyr Tyr Glu Asn Glu Thr Gly Phe Gly Ala Asn Val 485 490 495 Thr Leu Phe His Asn Arg Phe Arg Asn Lys Ile Asn Ser Val Ser Ile 500 505 510 Asp Asn Thr Thr Ser Thr Tyr Thr Asn Val Gly Lys Ala Ile Thr Gln 515 520 525 Gly Ile Glu Val Ala Ser Thr Ile Pro Leu Trp Ser Asp Asp Trp Met 530 535 540 Leu Gly Ile Asn Tyr Thr Phe Thr Asp Ser Glu Gln Lys Asp Gly Asn 545 550 555 560 Asn Lys Gly Ala Arg Leu Thr Asn Thr Pro Lys Asn Met Val Asn Ala 565 570 575 Arg Leu Asn Trp Asn Ile Asn Glu Gln Leu Ser Thr Trp Leu Lys Ala 580 585 590 Glu Tyr Arg Ser Lys Thr Ala Arg Phe Thr Gln Asn Tyr Ala Asn Leu 595 600 605 Ser Ala Ala Asn Lys Val Val Tyr Asn Asn Leu Gly Ser Glu Phe Lys 610 615 620 Pro Phe Ser Val Leu Asn Leu Gly Val Ala Tyr Lys Val Thr Lys Asp 625 630 635 640 Val Thr Leu Asn Gly Ala Val Asn Asn Leu Leu Asp Lys Asp Phe Thr 645 650 655 Arg Thr His Ile Phe Ala Val Gly Asn Gly Thr Thr Thr Ala Gly Asp 660 665 670 Tyr Phe Thr Ser Ser Gln Ser Thr Ala Gly Tyr Val Val Pro Gly Arg 675 680 685 Asn Tyr Trp Val Ser Val Asn Val Asn Phe 690 695 16673PRTYersinia pestis 16Met Lys Met Thr Arg Leu Tyr Pro Leu Ala Leu Gly Gly Leu Leu Leu 1 5 10 15 Pro Ala Ile Ala Asn Ala Gln Thr Ser Gln Gln Asp Glu Ser Thr Leu 20 25 30 Val Val Thr Ala Ser Lys Gln Ser Ser Arg Ser Ala Ser Ala Asn Asn 35 40 45 Val Ser Ser Thr Val Val Ser Ala Pro Glu Leu Ser Asp Ala Gly Val 50 55 60 Thr Ala Ser Asp Lys Leu Pro Arg Val Leu Pro Gly Leu Asn Ile Glu 65 70 75 80 Asn Ser Gly Asn Met Leu Phe Ser Thr Ile Ser Leu Arg Gly Val Ser 85 90 95 Ser Ala Gln Asp Phe Tyr Asn Pro Ala Val Thr Leu Tyr Val Asp Gly 100 105 110 Val Pro Gln Leu Ser Thr Asn Thr Ile Gln Ala Leu Thr Asp Val Gln 115 120 125 Ser Val Glu Leu Leu Arg Gly Pro Gln Gly Thr Leu Tyr Gly Lys Ser 130 135 140 Ala Gln Gly Gly Ile Ile Asn Ile Val Thr Gln Gln Pro Asp Ser Thr 145 150 155 160 Pro Arg Gly Tyr Ile Glu Gly Gly Val Ser Ser Arg Asp Ser Tyr Arg 165 170 175 Ser Lys Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly 180 185 190 Ser Val Thr Leu Leu Arg Gln Val Asp Asp Gly Asp Met Ile Asn Pro 195 200 205 Ala Thr Gly Ser Asp Asp Leu Gly Gly Thr Arg Ala Ser Ile Gly Asn 210 215 220 Val Lys Leu Arg Leu Ala Pro Asp Asp Gln Pro Trp Glu Met Gly Phe 225 230 235 240 Ala Ala Ser Arg Glu Cys Thr Arg Ala Thr Gln Asp Ala Tyr Val Gly 245 250 255 Trp Asn Asp Ile Lys Gly Arg Lys Leu Ser Ile Ser Asp Gly Ser Pro 260 265 270 Asp Pro Tyr Met Arg Arg Cys Thr Asp Ser Gln Thr Leu Ser Gly Lys 275 280 285 Tyr Thr Thr Asp Asp Trp Val Phe Asn Leu Ile Ser Ala Trp Gln Gln 290 295 300 Gln His Tyr Ser Arg Thr Phe Pro Ser Gly Ser Leu Ile Val Asn Met 305 310 315 320 Pro Gln Arg Trp Asn Gln Asp Val Gln Glu Leu Arg Ala Ala Thr Leu 325 330 335 Gly Asp Ala Arg Thr Val Asp Met Val Phe Gly Leu Tyr Arg Gln Asn 340 345 350 Thr Arg Glu Lys Leu Asn Ser Ala Tyr Asp Met Pro Thr Met Pro Tyr 355 360 365 Leu Ser Ser Thr Gly Tyr Thr Thr Ala Glu Thr Leu Ala Ala Tyr Ser 370 375 380 Asp Leu Thr Trp His Leu Thr Asp Arg Phe Asp Ile Gly Gly Gly Val 385 390 395 400 Arg Phe Ser His Asp Lys Ser Ser Thr Gln Tyr His Gly Ser Met Leu 405 410 415 Gly Asn Pro Phe Gly Asp Gln Gly Lys Ser Asn Asp Asp Gln Val Leu 420 425 430 Gly Gln Leu Ser Ala Gly Tyr Met Leu Thr Asp Asp Trp Arg Val Tyr 435 440 445 Thr Arg Val Ala Gln Gly Tyr Lys Pro Ser Gly Tyr Asn Ile Val Pro 450 455 460 Thr Ala Gly Leu Asp Ala Lys Pro Phe Val Ala Glu Lys Ser Ile Asn 465 470 475 480 Tyr Glu Leu Gly Thr Arg Tyr Glu Thr Ala Asp Val Thr Leu Gln Ala 485 490 495 Ala Thr Phe Tyr Thr His Thr Lys Asp Met Gln Leu Tyr Ser Gly Pro 500 505 510 Val Arg Met Gln Thr Leu Ser Asn Ala Gly Lys Ala Asp Ala Thr Gly 515 520 525 Val Glu Leu Glu Ala Lys Trp Arg Phe Ala Pro Gly Trp Ser Trp Asp 530 535 540 Ile Asn Gly Asn Val Ile Arg Ser Glu Phe Thr Asn Asp Ser Glu Leu 545 550 555 560 Tyr His Gly Asn Arg Val Pro Phe Val Pro Arg Tyr Gly Ala Gly Ser 565 570 575 Ser Val Asn Gly Val Ile Asp Thr Arg Tyr Gly Ala Leu Met Pro Arg 580 585 590 Leu Ala Val Asn Leu Val Gly Pro His Tyr Phe Asp Gly Asp Asn Gln 595 600 605 Leu Arg Gln Gly Thr Tyr Ala Thr Leu Asp Ser Ser Leu Gly Trp Gln 610 615 620 Ala Thr Glu Arg Met Asn Ile Ser Val Tyr Val Asp Asn Leu Phe Asp 625 630 635 640 Arg Arg Tyr Arg Thr Tyr Gly Tyr Met Asn Gly Ser Ser Ala Val Ala 645 650 655 Gln Val Asn Met Gly Arg Thr Val Gly Ile Asn Thr Arg Ile Asp Phe 660 665 670 Phe 17548PRTYersinia pseudotuberculosis 17Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Ile Lys Met 1 5 10 15 Leu Arg Gly Val Asn Ile Leu Ala Asp Ala Val Lys Val Thr Leu Gly 20 25 30 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ser Pro Thr 35 40 45 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp 50 55 60 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 65 70 75 80 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 85 90 95 Gln Ser Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn 100 105 110 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Ile Ala Ala Val 115 120 125 Glu Glu Leu Lys Lys Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile 130 135 140 Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Ser Thr Val Gly Glu 145 150 155 160 Leu Ile Ala Gln Ala Met Glu Lys Val Gly Lys Glu Gly Val Ile Thr 165 170 175 Val Glu Glu Gly Ser Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly 180 185 190 Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro 195 200 205 Glu Thr Gly Ser Ile Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp 210 215 220 Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val 225 230 235 240 Ala Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly 245 250 255 Glu Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys 260 265 270 Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280 285 Leu Gln Asp Ile Ala Thr Leu Thr Ala Gly Thr Val Ile Ser Glu Glu 290 295 300 Ile Gly Leu Glu Leu Glu Lys Thr Thr Leu Glu Asp Leu Gly Gln Ala 305 310 315 320 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Ile Ile Ile Asp Gly Val 325 330 335 Gly Asp Glu Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln 340 345 350 Ile Glu Asp Ala Thr Ser Asp Tyr Asp Lys Glu Lys Leu Gln Glu Arg 355 360 365 Val Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala 370 375 380 Thr Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu 385 390 395 400 His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly 405 410 415 Val Ala Leu Ile Arg Ala Ala His Ala Ile Ala Gly Leu Lys Gly Asp 420 425 430 Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu 435 440 445 Ser Pro Leu Arg Gln Ile Val Val Asn Ala Gly Glu Glu Ala Ser Val 450 455 460 Ile Ala Asn Lys Val Lys Ala Gly Glu Gly Ser Phe Gly Tyr Asn Ala 465 470 475 480 Tyr Thr Glu Glu Tyr Gly Asp Met Ile Ala Met Gly Ile Leu Asp Pro 485 490 495 Thr Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Ile Ala Gly 500 505 510 Leu Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Arg Asp Asp 515 520 525 Lys Gly Ala Asp Met Gly Ala Gly Gly Met Gly Gly Met Gly Gly Met 530 535 540 Gly Gly Met Met 545 18467PRTYersinia pestis 18Met Gln Met Lys Lys Leu Leu Pro Leu Leu Ile Gly Leu Ser Leu Ala 1 5 10 15 Gly Phe Ser Thr Met Ser Gln Ala Glu Asn Leu Leu Gln Val Tyr Lys 20 25 30 Gln Ala Arg Asp Ser Asn Pro Asp Leu Arg Lys Ala Ala Ala Asp Arg 35 40 45 Asp Ala Ala Tyr Glu Lys Ile Asn Glu Val Arg Ser Pro Leu Leu Pro 50 55 60 Gln Leu Gly Leu Ser Ala Gly Tyr Thr His Ala Asn Gly Phe Arg Asp 65 70 75 80 Ala Ser Asn Ser Pro Asp Ser Asn Ala Thr Ser Gly Ser Leu Lys Leu 85 90 95 Thr Gln Thr Ile Phe Asp Met Ser Lys Trp Arg Ala Leu Thr Leu Gln 100 105 110 Glu Lys Ala Ala Gly Ile Gln Asp Val Thr Phe Gln Thr Ser Glu Gln 115 120 125 Gln Leu Ile Leu Asn Thr Ala Thr Ala Tyr Phe Asn Val Leu Arg Ala 130 135 140 Ile Asp Ser Leu Ser Tyr Thr Glu Ala Gln Lys Gln Ser Val Tyr Arg 145 150 155 160 Gln Leu Asp Gln Thr Thr Gln Arg Phe Asn Val Gly Leu Val Ala Ile 165 170 175 Thr Asp Val Gln Asn Ala Arg Ala Ser Tyr Asp Thr Val Leu Ala Ala 180 185 190 Glu Val Ala Ala Arg Asn Asn Leu Asp Asn Ala Leu Glu Ser Leu Arg 195 200 205 Gln Ile Thr Gly Val Tyr Tyr Pro Glu Leu Ala Ser Leu Asn Val Glu 210 215 220 Arg Leu Lys Thr Gln Arg Pro Asp Ala Val Asn Asn Leu Leu Lys Glu 225 230 235 240 Ala Glu Lys Arg Asn Leu Ser Leu Leu Ser Ala Arg Leu Ser Gln Asp 245 250 255 Leu Ala Arg Glu Gln Ile Lys Ser Ala Glu Thr Gly Tyr Met Pro Thr 260 265 270 Val Asp Leu Thr Ala Ser Ser Ser Ile Thr Asn Thr Arg Tyr Ser Gly 275 280 285 Gly Thr Pro Ser Ser Gln Gln Val Asn Asn Asp Ser Gly Gln Asn Gln 290 295 300 Ile Gly Val Gln Phe Ser Leu Pro Leu Tyr Ser Gly Gly Ala Thr Asn 305 310 315 320 Ser Ala Val Lys Gln Ala Gln Tyr Asn Phe Val Gly Ala Ser Glu Leu 325 330 335 Leu Glu Ser Ala His Arg Asn Met Val Gln Thr Leu Arg Ser Ser Phe 340 345 350 Asn Asn Ile Ser Ala Ser Ile Ser Ser Ile Asn Ala Tyr Gln Gln Val 355 360 365 Val Ile Ser Asn Gln Ser Ser Leu Asp Ala Met Glu Ala Gly Tyr Gln 370 375 380 Val Gly Thr Arg Thr Ile Leu Asp Val Leu Thr Ala Thr Thr Asn Leu 385 390 395 400 Tyr Gln Ser Lys Gln Gln Leu Ala Asp Ala Arg Tyr Asn Tyr Leu Ile 405 410 415 Asn Gln Leu Asn Ile Lys Ser Ala Leu Gly Thr Leu Asn Met Asn Asp 420 425 430 Leu Met Ala Leu Asn Ala Val Leu Asp Lys Pro Val Pro Thr Ser Ala 435 440 445 Ala Ala Leu Ala Pro Glu Asn Thr Thr Arg Gln Thr Val Thr Thr Pro 450 455 460 Arg Ala Gln 465 19394PRTYersinia pseudotuberculosis 19Met Ser Lys Glu Lys Phe Glu Arg Thr Lys Pro His Val Asn Val Gly 1 5 10 15 Thr Ile Gly His Val Asp His Gly Lys Thr Thr Leu Thr Ala Ala Ile 20 25 30 Thr Thr Val Leu Ala Lys Thr Tyr Gly Gly Ser Ala Arg Ala Phe Asp 35 40 45 Gln Ile Asp Asn Ala Pro Glu Glu Lys Ala Arg Gly Ile Thr Ile Asn 50 55 60 Thr Ser His Val Glu Tyr Asp Thr Pro Ala Arg His Tyr Ala His Val 65 70 75 80 Asp Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met Ile Thr Gly Ala 85 90 95 Ala Gln Met Asp Gly Ala Ile Leu Val Val Ala Ala Thr Asp Gly Pro 100 105 110 Met Pro Gln Thr Arg Glu His Ile Leu Leu Gly Arg Gln Val Gly Val 115 120 125 Pro Tyr Ile Ile Val Phe Met Asn Lys Cys Asp Met Val Asp Asp Glu 130 135 140 Glu Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala 145 150 155 160 Tyr Asp Phe Pro Gly Asp Asp Leu Pro Val Val Arg Gly Ser Ala Leu 165 170 175 Lys Ala Leu Glu Gly Glu Ala Glu Trp Glu Ala Lys Ile Ile Glu Leu 180 185 190 Ala Gly Tyr Leu Asp Ser Tyr Ile Pro Glu Pro Glu Arg Ala Ile Asp 195 200 205 Lys Pro Phe Leu Leu Pro Ile Glu Asp Val Phe Ser Ile Ser Gly Arg 210 215 220 Gly Thr Val Val Thr Gly Arg Val Glu Arg Gly Ile Val Lys Val Gly 225 230 235 240 Glu Glu Val Glu Ile Val Gly Ile Lys Asp Thr Val Lys Ser Thr Cys 245 250 255 Thr Gly Val Glu Met Phe Arg Lys Leu Leu Asp Glu Gly Arg Ala Gly 260 265 270 Glu Asn Val Gly Val Leu Leu Arg Gly Ile Lys Arg Glu Asp Ile Glu 275 280 285 Arg Gly Gln Val Leu Ala Lys Pro Gly Ser Ile Lys Pro His Thr Thr 290 295

300 Phe Glu Ser Glu Val Tyr Ile Leu Ser Lys Asp Glu Gly Gly Arg His 305 310 315 320 Thr Pro Phe Phe Lys Gly Tyr Arg Pro Gln Phe Tyr Phe Arg Thr Thr 325 330 335 Asp Val Thr Gly Thr Ile Glu Leu Pro Glu Gly Val Glu Met Val Met 340 345 350 Pro Gly Asp Asn Ile Asn Met Ile Val Thr Leu Ile His Pro Ile Ala 355 360 365 Met Asp Asp Gly Leu Arg Phe Ala Ile Arg Glu Gly Gly Arg Thr Val 370 375 380 Gly Ala Gly Val Val Ala Lys Val Ile Ala 385 390 20371PRTYersinia pestis 20Met Lys Leu Arg Val Leu Ser Phe Ile Ile Pro Ala Leu Leu Val Ala 1 5 10 15 Gly Ser Ala Ser Ala Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys Leu 20 25 30 Asp Leu Tyr Gly Lys Ile Asp Gly Leu His Tyr Phe Ser Asp Asn Lys 35 40 45 Asn Leu Asp Gly Asp Gln Ser Tyr Met Arg Phe Gly Leu Lys Gly Glu 50 55 60 Thr Gln Ile Thr Asp Gln Leu Thr Gly Tyr Gly Gln Trp Glu Tyr Gln 65 70 75 80 Val Asn Leu Asn Lys Ala Glu Asn Glu Asp Gly Asn His Asp Ser Phe 85 90 95 Thr Arg Val Gly Phe Ala Gly Leu Lys Phe Ala Asp Tyr Gly Ser Leu 100 105 110 Asp Tyr Gly Arg Asn Tyr Gly Val Leu Tyr Asp Val Thr Ser Trp Thr 115 120 125 Asp Val Leu Pro Glu Phe Gly Gly Asp Thr Tyr Gly Ala Asp Asn Phe 130 135 140 Leu Ser Gln Arg Gly Asn Gly Met Leu Thr Tyr Arg Asn Thr Asn Phe 145 150 155 160 Phe Gly Leu Val Asp Gly Leu Asn Phe Ala Leu Gln Tyr Gln Gly Lys 165 170 175 Asn Gly Ser Ser Ser Glu Thr Asn Asn Gly Arg Gly Val Ala Asp Gln 180 185 190 Asn Gly Asp Gly Tyr Gly Met Ser Leu Ser Tyr Asp Leu Gly Trp Gly 195 200 205 Val Ser Ala Ser Ala Ala Met Ala Ser Ser Leu Arg Thr Thr Ala Gln 210 215 220 Asn Asp Leu Gln Tyr Gly Gln Gly Lys Arg Ala Asn Ala Tyr Thr Gly 225 230 235 240 Gly Leu Lys Tyr Asp Ala Asn Asn Val Tyr Leu Ala Ala Asn Tyr Thr 245 250 255 Gln Thr Tyr Asn Leu Thr Arg Phe Gly Asp Phe Ser Asn Arg Ser Ser 260 265 270 Asp Ala Ala Phe Gly Phe Ala Asp Lys Ala His Asn Ile Glu Val Val 275 280 285 Ala Gln Tyr Gln Phe Asp Phe Gly Leu Arg Pro Ser Val Ala Tyr Leu 290 295 300 Gln Ser Lys Gly Lys Asp Ile Gly Ile Tyr Gly Asp Gln Asp Leu Leu 305 310 315 320 Lys Tyr Val Asp Ile Gly Ala Thr Tyr Phe Phe Asn Lys Asn Met Ser 325 330 335 Thr Tyr Val Asp Tyr Lys Ile Asn Leu Leu Asp Lys Asn Asp Phe Thr 340 345 350 Lys Asn Ala Arg Ile Asn Thr Asp Asp Ile Val Ala Val Gly Met Val 355 360 365 Tyr Gln Phe 370 21254PRTYersinia pestis 21Met Tyr Asn Ile Asp Tyr Asn Ser Phe Arg Ser Val Lys Gly Phe Asn 1 5 10 15 Arg Arg Val Arg Phe Leu Val Met His Tyr Thr Ala Phe Asn Phe Lys 20 25 30 Asp Ser Ile Asp Ala Leu Thr Gly Pro Ser Val Ser Ala His Tyr Leu 35 40 45 Val Pro Asp Pro Thr Glu Gln Thr Tyr Ile Asp Ala Gly Phe Lys Asp 50 55 60 Met Arg Ile Phe Asn Leu Val Asp Glu Asn Glu Arg Ala Trp His Ala 65 70 75 80 Gly Val Ser Tyr Trp Asp Gly Arg Asn Asn Leu Asn Asp Thr Ala Ile 85 90 95 Gly Ile Glu Thr Val Asn Leu Ala Thr Asp Asn Asp Gly Val Phe Thr 100 105 110 Phe Pro Pro Tyr Asn Val Thr Gln Ile Ala Ala Ile Lys Ala Leu Ala 115 120 125 Ser Asn Ile Leu Tyr Arg Phe Pro Asp Ile Thr Pro Val Asn Val Val 130 135 140 Gly His Ser Asp Ile Ala Pro Gly Arg Lys Ser Asp Pro Gly Ala Ala 145 150 155 160 Phe Pro Trp Lys Ala Leu Tyr Asp Ala Gly Ile Gly Ala Trp Tyr Asp 165 170 175 Asp Glu Thr Lys Gln Arg Tyr Leu Asp Gln Phe Leu Cys Ser Leu Pro 180 185 190 Ser Lys Asn Asp Ile Ile Ser Lys Leu Lys Arg Tyr Gly Tyr Asp Thr 195 200 205 Ser Gly Ala Val Ser Glu Val Gly Tyr Asn Gln Leu Ile Arg Ala Phe 210 215 220 Gln Leu His Phe Arg Pro Cys Asn Tyr Asp Gly Ile Pro Asp Ala Glu 225 230 235 240 Thr Val Ala Ile Leu Tyr Ala Leu Val Asp Lys Tyr Lys Pro 245 250 22283PRTYersinia pestis 22 Met Arg Lys Leu Leu Ser Gly Gly Leu Leu Leu Leu Leu Ala Gly Cys 1 5 10 15 Ser Ser Ser Asp His Arg Asn Ser Asn Glu Leu Ile Asp Arg Gly Thr 20 25 30 Tyr Gln Ile Asp Thr His Tyr Pro Ser Val Ala Lys Asn Glu Arg Val 35 40 45 Arg Phe Leu Val Leu His Tyr Thr Ala Val Gly Asp Ala Glu Ser Leu 50 55 60 Arg Leu Leu Thr Gln Gly Glu Val Ser Ala His Tyr Leu Ile Pro Thr 65 70 75 80 His Pro Lys Lys Ala Gly Gly Lys Ala Ile Ala Leu Gln Leu Val Pro 85 90 95 Glu Ala Gln Arg Ala Trp His Ala Gly Val Ser Ser Trp Gln Gly Arg 100 105 110 Asn Asn Leu Asn Asp Thr Ser Ile Gly Ile Glu Ile Val Asn Leu Gly 115 120 125 Phe Thr Glu Lys Met Leu Gly Arg Thr Trp Tyr Pro Tyr Asn Glu Ser 130 135 140 Gln Ile Glu Leu Ile Glu Gln Leu Thr Lys Asp Ile Val Gln Arg Tyr 145 150 155 160 Asn Ile Ser Pro Ser Asp Val Val Ala His Ser Asp Ile Ala Pro Leu 165 170 175 Arg Lys Ser Asp Pro Gly Pro Leu Phe Pro Trp Lys Arg Leu Ala Glu 180 185 190 Lys Gly Val Gly Ala Trp Pro Asp Asp Ala Thr Val Ala Lys Tyr Ile 195 200 205 Gly Gly Arg Asp Lys Lys Gly Ala Ala Ser Val Ala Val Ile Gln Gln 210 215 220 Ala Leu Ala Ala Tyr Gly Tyr Lys Ile Pro Gln Asn Gly Gln Leu Asp 225 230 235 240 Thr Glu Thr Arg Gln Val Ile Lys Ala Phe Gln Met His Phe Arg Pro 245 250 255 Gln Asp Phe Ser Gly Val Pro Asp Val Glu Thr Glu Ala Ile Ala Leu 260 265 270 Ala Leu Val Glu Lys Tyr Arg Thr Leu Ser Thr 275 280 23194PRTYersinia pestis 23Met Val Thr Val Leu Gly Ile Val Ile Thr Ile Trp Met Val Phe Met 1 5 10 15 Asn Lys Thr Leu Leu Val Ser Ser Leu Ile Ala Cys Leu Ser Ile Ala 20 25 30 Ser Val Asn Val Tyr Ala Glu Gly Glu Ser Ser Ile Ser Ile Gly Tyr 35 40 45 Ala Gln Ser Arg Val Lys Glu Asp Gly Tyr Lys Leu Asp Lys Asn Pro 50 55 60 Arg Gly Phe Asn Leu Lys Tyr Arg Tyr Glu Phe Asn Asn Asp Trp Gly 65 70 75 80 Val Ile Gly Ser Phe Ala Gln Thr Arg Arg Gly Phe Glu Glu Ser Val 85 90 95 Asp Gly Phe Lys Leu Ile Asp Gly Asp Phe Lys Tyr Tyr Ser Val Thr 100 105 110 Ala Gly Pro Val Phe Arg Ile Asn Glu Tyr Val Ser Leu Tyr Gly Leu 115 120 125 Leu Gly Ala Gly His Gly Lys Ala Lys Phe Ser Ser Ile Phe Gly Gln 130 135 140 Ser Glu Ser Arg Ser Lys Thr Ser Leu Ala Tyr Gly Ala Gly Leu Gln 145 150 155 160 Phe Asn Pro His Pro Asn Phe Val Ile Asp Ala Ser Tyr Glu Tyr Ser 165 170 175 Lys Leu Asp Asp Val Lys Val Gly Thr Trp Met Leu Gly Ala Gly Tyr 180 185 190 Arg Phe 247PRTYersinia enterocolitica 24Phe His Gln Leu Asp Asn Arg 1 5 2511PRTYersinia enterocolitica 25Val Asn Phe Thr Ala Gly Val Gly Gly Tyr Arg 1 5 10 2612PRTYersinia enterocolitica 26Asn Ser Val Ser Ile Gly His Glu Ser Leu Asn Arg 1 5 10 2715PRTYersinia enterocolitica 27Ala Ser Thr Ser Asp Thr Gly Val Ala Val Gly Phe Asn Ser Lys 1 5 10 15 2815PRTYersinia enterocolitica 28Ser Ala Glu Thr Leu Ala Ser Ala Asn Val Tyr Ala Asp Ser Lys 1 5 10 15 2914PRTYersinia enterocoliticaMOD_RES(12)..(12)Oxidized Met 29Glu Ala Phe Asp Leu Ser Asn Asp Ala Leu Asp Met Ala Lys 1 5 10 3015PRTYersinia enterocolitica 30Ser Ala Glu Val Leu Gly Ile Ala Asn Asn Tyr Thr Asp Ser Lys 1 5 10 15 3117PRTYersinia enterocolitica 31Ala Leu Gly Asp Ser Ala Val Thr Tyr Gly Ala Gly Ser Thr Ala Gln 1 5 10 15 Lys 3215PRTYersinia enterocoliticaMOD_RES(12)..(12)Oxidized Met 32Glu Ala Phe Asp Leu Ser Asn Asp Ala Leu Asp Met Ala Lys Lys 1 5 10 15 3324PRTYersinia enterocolitica 33Ala Ala Val Ala Val Gly Ala Gly Ser Ile Ala Thr Gly Val Asn Ser 1 5 10 15 Val Ala Ile Gly Pro Leu Ser Lys 20 346PRTYersinia enterocolitica 34Asp Ile Gly Asn Ile Arg 1 5 357PRTYersinia enterocolitica 35Phe Phe Val Ser Tyr Gln Trp 1 5 369PRTYersinia enterocolitica 36Val Asn Gly Gln Asp Val Thr Leu Arg 1 5 379PRTYersinia enterocolitica 37Ala Ser Tyr Phe Asp Thr Asn Ala Lys 1 5 3811PRTYersinia enterocolitica 38Asp Leu Pro Val Ser Ile Leu Ala Gly Thr Arg 1 5 10 3911PRTYersinia enterocolitica 39Gln Gly Val Leu Thr Leu Val Asp Gly Ile Arg 1 5 10 4012PRTYersinia enterocolitica 40Asn Ile Pro Gly Leu Thr Val Thr Gly Ser Gly Arg 1 5 10 4110PRTYersinia enterocolitica 41Tyr Tyr Asn Asn Ser Ala Leu Glu Pro Lys 1 5 10 4212PRTYersinia enterocolitica 42Ala Pro Thr Met Gly Glu Met Tyr Asn Asp Ser Lys 1 5 10 4312PRTYersinia enterocolitica 43Ile Asp Gln Ile Gln Ser Leu Ser Ala Asn Leu Arg 1 5 10 4413PRTYersinia enterocolitica 44Thr Asp Asp Val Asp Gly Ile Leu Ser Phe Gly Thr Arg 1 5 10 4514PRTYersinia enterocolitica 45Gly Met Thr Thr Thr Val Val Leu Gly Asn Ala Phe Asp Lys 1 5 10 4614PRTYersinia enterocolitica 46Ile Ala Asp Thr Met Val Val Thr Ala Thr Gly Asn Glu Arg 1 5 10 4713PRTYersinia enterocolitica 47Phe Gly Ser Gly Trp Leu Gln Asp Glu Ile Thr Leu Arg 1 5 10 4816PRTYersinia enterocolitica 48Asn Pro Gln Thr Ser Ala Ala Ser Ser Thr Asn Leu Met Thr Asp Arg 1 5 10 15 4914PRTYersinia enterocolitica 49Phe Asn Asp Leu Met Met Ala Glu Asp Asp Leu Gln Phe Lys 1 5 10 5017PRTYersinia enterocolitica 50Gly Ser Ser Glu Gly Tyr Ala Asp Val Asp Ala Asp Lys Trp Ser Ser 1 5 10 15 Arg 5118PRTYersinia enterocolitica 51Gln Glu Gln Thr Pro Ser Gly Ala Thr Glu Ser Phe Pro Gln Ala Asp 1 5 10 15 Ile Arg 5219PRTYersinia enterocolitica 52Gln Gly Thr Asp Thr Gly His Leu Asn Ser Thr Phe Leu Asp Pro Ala 1 5 10 15 Leu Val Lys 5319PRTYersinia enterocolitica 53Gln Ser Asp Gly Phe Asn Ala Pro Asn Asp Glu Thr Ile Ser Asn Val 1 5 10 15 Leu Ala Lys 5421PRTYersinia enterocolitica 54Val Tyr Ser Ala Ala Ala Thr Gly Asp His Ser Phe Gly Leu Gly Ala 1 5 10 15 Ser Ala Phe Gly Arg 20 5519PRTYersinia enterocolitica 55Leu Phe Thr Asp Ser Phe Ala Ser His Leu Leu Thr Tyr Gly Thr Glu 1 5 10 15 Ala Tyr Lys 5621PRTYersinia enterocolitica 56Val Ser Ser Ser Gly Thr Pro Gln Ala Gly Tyr Gly Val Asn Asp Phe 1 5 10 15 Tyr Val Ser Tyr Lys 20 5722PRTYersinia enterocolitica 57Gly Ala Val Ser Val Thr Pro Thr Asp Trp Leu Met Leu Phe Gly Ser 1 5 10 15 Tyr Ala Gln Ala Phe Arg 20 5830PRTYersinia enterocolitica 58Ser Ser Phe Glu Ala Pro Met Met Val Thr Val Val Glu Ala Asp Thr 1 5 10 15 Pro Thr Ser Glu Thr Ala Thr Ser Ala Thr Asp Met Leu Arg 20 25 30 599PRTYersinia enterocolitica 59Asn Asp Ala Ser Val Gln Asn Val Arg 1 5 6010PRTYersinia enterocolitica 60Ile Gly Phe Leu Gly Gln Gln Asp Ala Arg 1 5 10 6110PRTYersinia enterocolitica 61Val Asn Leu Gly Tyr Ala Ala Asn Tyr Arg 1 5 10 6210PRTYersinia enterocolitica 62Gly Tyr Gly Asn Pro Ser Gln Asn Tyr Arg 1 5 10 6310PRTYersinia enterocolitica 63Tyr Gly Asp Asp Asp Gln Phe Gly Val Arg 1 5 10 6411PRTYersinia enterocolitica 64Gly His Phe Asp Thr Gly Pro Ile Thr His Lys 1 5 10 6511PRTYersinia enterocolitica 65Leu Leu Ala Ser Ala Thr Trp Leu Asp Pro Lys 1 5 10 6612PRTYersinia enterocolitica 66Asn Val Pro Phe Asn Val Ile Gly Tyr Thr Ser Lys 1 5 10 6712PRTYersinia enterocolitica 67Leu Lys Pro Trp Thr Arg Leu Asp Leu Gly Val Arg 1 5 10 6815PRTYersinia enterocolitica 68Val Ser Leu Tyr Ala Asn His Ile Glu Ala Leu Gly Pro Gly Lys 1 5 10 15 6915PRTYersinia enterocolitica 69Gly Ile Glu Leu Asn Val Phe Gly Glu Pro Val Phe Gly Thr Arg 1 5 10 15 7015PRTYersinia enterocolitica 70Thr Asn Asp Thr Ile Thr Val Val Gly Ala Gln Glu Thr Phe Arg 1 5 10 15 7114PRTYersinia enterocolitica 71Val Thr Pro Ile Tyr Gly Ile Met Val Lys Pro Trp Glu Lys 1 5 10 7217PRTYersinia enterocolitica 72Asn Phe Asp Ser Gly Val Pro Asn Ser Ala Gly Ser Leu Asp Ala Met 1 5 10 15 Lys 7317PRTYersinia enterocolitica 73Leu Tyr Val Pro Tyr Val Ala Asp Ser Val Ala Gly Leu Gly Gly Ile 1 5 10 15 Arg 7419PRTYersinia enterocolitica 74Val Thr Val Asp Tyr Gly Ser Ala Ser Gln Val Gly Gly Ala Leu Asp 1 5 10 15 Val Gly Arg 7520PRTYersinia enterocolitica 75Ala Gly Gly Asn Asp Leu Ile Pro Thr Tyr Leu Asp Gly Gln Val Ala 1 5 10 15 Asn Gly Gly Arg 20 7619PRTYersinia enterocolitica 76Ser Glu Tyr Asp Val Ser Gln Asn Trp Thr Val Tyr Gly Ser Val Gly 1 5 10 15 Ala Ser Arg 7720PRTYersinia enterocolitica 77Gly Tyr Asn Leu Asp Gly Asp Asp Ile Ser Phe Gly Gly Leu Phe Gly 1 5 10 15 Val Leu Pro Arg 20 7819PRTYersinia enterocolitica 78Ser Gly Ser Gln Tyr Ala Asn Glu Ala Asn Thr Leu Lys Leu Lys Pro 1 5 10 15 Trp Thr Arg 7925PRTYersinia enterocolitica 79Gly Ala Asn Ala Phe Ile Asn Gly Ile Ser Pro Ser Gly Ser Gly Val 1 5 10 15 Gly Gly Met Ile Asn Leu Glu Pro Lys 20 25 8023PRTYersinia enterocolitica 80Asn Glu Glu Thr Gly Gln Tyr Gly Ala Pro Met Leu Thr Asn Asn Asn 1 5 10 15 Gly Asp Ala Thr

Ile Ser Arg 20 8124PRTYersinia enterocolitica 81Ser Ala Pro Tyr Gln Tyr Asn Gly Lys Pro Val Val Asn Ala Gly Gln 1 5 10 15 Ile Pro Gly Ile Ile His Ser Lys 20 8231PRTYersinia enterocolitica 82Tyr Gly Gly Thr Leu Ala Leu Phe Glu Ile Thr Arg Pro Thr Gly Met 1 5 10 15 Val Asp Pro Ala Thr Asn Val Tyr Gly Phe Tyr Gly Glu Gln Arg 20 25 30 837PRTYersinia enterocolitica 83Tyr Asp Thr Val Ala Leu Arg 1 5 849PRTYersinia enterocolitica 84Val Leu Leu Gly Val Asp Phe Gln Lys 1 5 858PRTYersinia enterocolitica 85Phe Asp Asp Val Trp Ser Phe Arg 1 5 8610PRTYersinia enterocolitica 86Ser Val Gln Ala Thr Val Gly Tyr Asp Phe 1 5 10 8711PRTYersinia enterocolitica 87Ala Asp Leu Gly Thr Trp Ala Ala Ser Leu Lys 1 5 10 8810PRTYersinia enterocolitica 88Gln Trp Ala Asp Asp Ala Asn Thr Leu Arg 1 5 10 8911PRTYersinia enterocolitica 89Val Asn Ser Gln Gly Leu Glu Leu Glu Ala Arg 1 5 10 9012PRTYersinia enterocolitica 90Ala Val Pro Ala Thr Tyr Tyr Val Pro Ala Gly Lys 1 5 10 9111PRTYersinia enterocolitica 91Leu Ser Val Ile Ala Gly Tyr Thr Tyr Asn Arg 1 5 10 9212PRTYersinia enterocolitica 92Val Pro Ser Tyr Thr Leu Gly Asp Ala Ser Val Arg 1 5 10 9311PRTYersinia enterocolitica 93Arg Pro Gln Phe Thr Ser Glu Gly His Phe Arg 1 5 10 9413PRTYersinia enterocolitica 94Gly Phe Phe Asp Gly Glu Ser Asn His Asn Val Phe Lys 1 5 10 9514PRTYersinia enterocolitica 95Gly Ala Phe Val Gln Leu Asn Val Asn Asn Ile Ala Asp Lys 1 5 10 9612PRTYersinia enterocolitica 96Trp Gln Gln Ile Tyr Ser Tyr Glu Phe Ser His Lys 1 5 10 9714PRTYersinia enterocolitica 97Gly Phe Phe Asp Gly Glu Ser Asn His Asn Val Phe Lys Arg 1 5 10 9816PRTYersinia enterocolitica 98Gly Phe His Gly Gly Asp Val Asn Asn Thr Phe Leu Asp Gly Leu Arg 1 5 10 15 9913PRTYersinia enterocolitica 99Arg Trp Gln Gln Ile Tyr Ser Tyr Glu Phe Ser His Lys 1 5 10 10018PRTYersinia enterocolitica 100Ala Gly His Glu Ala Asp Leu Pro Thr Ser Gly Tyr Thr Ala Thr Thr 1 5 10 15 Thr Lys 10117PRTYersinia enterocolitica 101Thr Asp Gln Pro Leu Ile Leu Thr Ala Gln Ser Val Ser Val Val Thr 1 5 10 15 Arg 10220PRTYersinia enterocolitica 102Asp Pro Ser Gly Gly Tyr His Ser Ala Val Pro Ala Asp Gly Ser Ile 1 5 10 15 Tyr Gly Gln Lys 20 10321PRTYersinia enterocolitica 103Gly Pro Ser Ser Ala Leu Tyr Gly Gln Ser Ile Pro Gly Gly Val Val 1 5 10 15 Met Met Thr Ser Lys 20 10418PRTYersinia enterocolitica 104Lys Tyr Val Ala Ala Cys Tyr Ser Thr Ser Tyr Cys Tyr Trp Gly Ala 1 5 10 15 Glu Arg 10520PRTYersinia enterocolitica 105Tyr Ala Ile Ala Pro Ser Leu Leu Trp Gln Pro Asp Glu Asn Thr Ser 1 5 10 15 Leu Leu Leu Arg 20 10620PRTYersinia enterocolitica 106Leu Leu Ser Asp Gly Gly Ser Tyr Asn Val Leu Gln Val Asp Pro Trp 1 5 10 15 Phe Leu Glu Arg 20 10724PRTYersinia enterocolitica 107Gln Asn Ala Ser Tyr Thr His Ser Asn Thr Gln Leu Glu Gln Val Tyr 1 5 10 15 Gln Gly Gly Trp Asn Ser Asp Arg 20 10826PRTYersinia enterocolitica 108Leu Thr Ala Gly Asn Asn Asn Thr Gln Val Ala Ala Phe Asp Tyr Thr 1 5 10 15 Asp Ala Ile Ser Glu His Trp Ala Phe Arg 20 25 10925PRTYersinia enterocolitica 109Arg Tyr Glu Gln Ser Gly Val Tyr Leu Gln Asp Glu Met Thr Leu Asp 1 5 10 15 Asn Trp His Leu Asn Leu Ser Gly Arg 20 25 11031PRTYersinia enterocolitica 110Gln Gln Met Asp Asp Gln Asn Val Ala Thr Val Asn Gln Ala Leu Asn 1 5 10 15 Tyr Thr Pro Gly Val Phe Thr Gly Phe Ser Gly Gly Ala Thr Arg 20 25 30 1116PRTYersinia enterocolitica 111Val Pro Phe Val Pro Arg 1 5 1127PRTYersinia enterocolitica 112Thr Val Gly Ile Asn Thr Arg 1 5 1137PRTYersinia enterocolitica 113Tyr Gly Ala Leu Met Pro Arg 1 5 1148PRTYersinia enterocolitica 114Phe Asp Ile Gly Gly Gly Val Arg 1 5 1159PRTYersinia enterocolitica 115Gly Pro Gln Gly Thr Leu Tyr Gly Lys 1 5 11610PRTYersinia enterocolitica 116Gly Tyr Ile Glu Gly Gly Val Ser Ser Arg 1 5 10 1179PRTYersinia enterocolitica 117Ser Ile Asn Tyr Glu Leu Gly Thr Arg 1 5 1189PRTYersinia enterocolitica 118Trp Asn Gln Asp Val Gln Glu Leu Arg 1 5 11910PRTYersinia enterocolitica 119Thr Val Asp Met Val Phe Gly Leu Tyr Arg 1 5 10 12014PRTYersinia enterocolitica 120Tyr Gly Ala Gly Ser Ser Val Asn Gly Val Ile Asp Thr Arg 1 5 10 12113PRTYersinia enterocolitica 121Leu Ser Leu Ser Asp Gly Ser Pro Asp Pro Tyr Met Arg 1 5 10 12213PRTYersinia enterocolitica 122Ala Thr Gln Asp Ala Tyr Val Gly Trp Asn Asp Ile Lys 1 5 10 12313PRTYersinia enterocolitica 123Ile Asn Ile Ser Val His Val Asp Asn Leu Phe Asp Arg 1 5 10 12414PRTYersinia enterocolitica 124Thr Phe Pro Ser Gly Ser Leu Ile Val Asn Met Pro Gln Arg 1 5 10 12514PRTYersinia enterocolitica 125Lys Leu Ser Leu Ser Asp Gly Ser Pro Asp Pro Tyr Met Arg 1 5 10 12614PRTYersinia enterocolitica 126Ser Glu Phe Thr Asn Asp Ser Glu Leu Tyr His Gly Asn Arg 1 5 10 12715PRTYersinia enterocolitica 127Phe Ala Pro Gly Trp Ser Trp Asp Ile Asn Gly Asn Val Ile Arg 1 5 10 15 12816PRTYersinia enterocolitica 128Leu Ala Pro Asp Asp Gln Pro Trp Glu Met Gly Phe Ala Ala Ser Arg 1 5 10 15 12918PRTYersinia enterocolitica 129Thr Tyr Gly Tyr Met Asn Gly Ser Ser Ala Val Ala Gln Val Asn Met 1 5 10 15 Gly Arg 13019PRTYersinia enterocolitica 130Ser Ala Gln Gly Gly Ile Ile Asn Ile Val Thr Gln Gln Pro Asp Ser 1 5 10 15 Thr Pro Arg 13118PRTYersinia enterocolitica 131Gln Gly Thr Tyr Ala Thr Leu Asp Ser Ser Leu Gly Trp Gln Ala Thr 1 5 10 15 Glu Arg 13219PRTYersinia enterocolitica 132Asp Met Gln Leu Tyr Ser Gly Pro Val Gly Met Gln Thr Leu Ser Asn 1 5 10 15 Ala Gly Lys 13319PRTYersinia enterocolitica 133Ser Ser Thr Gln Tyr His Gly Ser Met Leu Gly Asn Pro Phe Gly Asp 1 5 10 15 Gln Gly Lys 13418PRTYersinia enterocolitica 134Leu Ala Val Asn Leu Val Gly Pro His Tyr Phe Asp Gly Asp Asn Gln 1 5 10 15 Leu Arg 13518PRTYersinia enterocolitica 135Leu Arg Leu Ala Pro Asp Asp Gln Pro Trp Glu Met Gly Phe Ala Ala 1 5 10 15 Ser Arg 13621PRTYersinia enterocolitica 136Gln Val Asp Asp Gly Asp Met Ile Asn Pro Ala Thr Gly Ser Asp Asp 1 5 10 15 Leu Gly Gly Thr Arg 20 13720PRTYersinia enterocolitica 137Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly Ser Val 1 5 10 15 Thr Leu Leu Arg 20 13821PRTYersinia enterocolitica 138Val Leu Pro Gly Leu Asn Ile Glu Asn Ser Gly Asn Met Leu Phe Ser 1 5 10 15 Thr Ile Ser Leu Arg 20 13920PRTYersinia enterocolitica 139Ser Glu Phe Thr Asn Asp Ser Glu Leu Tyr His Gly Asn Arg Val Pro 1 5 10 15 Phe Val Pro Arg 20 14022PRTYersinia enterocolitica 140Ser Lys Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly 1 5 10 15 Ser Val Thr Leu Leu Arg 20 14121PRTYersinia enterocolitica 141Ser Asn Asp Asp Gln Val Leu Gly Gln Leu Ser Ala Gly Tyr Met Leu 1 5 10 15 Thr Asp Asp Trp Arg 20 14227PRTYersinia enterocolitica 142Ser Ala Ser Ala Asn Asn Val Ser Ser Thr Val Val Ser Ala Pro Glu 1 5 10 15 Leu Ser Asp Ala Gly Val Thr Ala Ser Asp Lys 20 25 14321PRTYersinia enterocolitica 143Tyr Thr Thr Asp Asp Trp Val Phe Asn Leu Ile Ser Ala Trp Gln Gln 1 5 10 15 Gln His Tyr Ser Arg 20 14427PRTYersinia enterocolitica 144Ile Ala Gln Gly Tyr Lys Pro Ser Gly Tyr Asn Ile Val Pro Thr Ala 1 5 10 15 Gly Leu Asp Ala Lys Pro Phe Val Ala Glu Lys 20 25 14530PRTYersinia enterocolitica 145Ser Ala Ser Ala Asn Asn Val Ser Ser Thr Val Val Ser Ala Pro Glu 1 5 10 15 Leu Ser Asp Ala Gly Val Thr Ala Ser Asp Lys Leu Pro Arg 20 25 30 1468PRTYersinia enterocolitica 146Val Ser Gly Leu Leu Ser His Arg 1 5 1477PRTYersinia enterocolitica 147Thr Ser Glu Tyr Leu Asn Arg 1 5 1487PRTYersinia enterocolitica 148Glu Trp His Gly Thr Val Arg 1 5 1499PRTYersinia enterocolitica 149Tyr Thr Leu Ile Leu Val Asp Gly Lys 1 5 1509PRTYersinia enterocolitica 150Arg Val Asp Ile Glu Val Asn Asp Lys 1 5 15110PRTYersinia enterocolitica 151Val Gly Lys Glu Trp His Gly Thr Val Arg 1 5 10 15210PRTYersinia enterocolitica 152Tyr Thr Leu Ile Leu Val Asp Gly Lys Arg 1 5 10 15311PRTYersinia enterocolitica 153Leu Met Gly Gly Val Tyr Asn Val Leu Asp Lys 1 5 10 15413PRTYersinia enterocolitica 154Ile Gln Asp Ser Ala Ala Ser Ile Ser Val Val Thr Arg 1 5 10 15514PRTYersinia enterocolitica 155Met Asp Gln Asp Glu Asn Tyr Gly Thr His Trp Thr Pro Arg 1 5 10 15614PRTYersinia enterocolitica 156Asn Glu Phe Asp Phe Asp Ile Gly His Tyr Val Gln Asp Arg 1 5 10 15719PRTYersinia enterocolitica 157Asp Val Pro Gly Val Val Val Thr Gly Gly Gly Ser His Ser Asp Ile 1 5 10 15 Ser Ile Arg 15824PRTYersinia enterocolitica 158Gly Thr Arg Pro Asn Ser Asp Gly Ser Gly Ile Glu Gln Gly Trp Leu 1 5 10 15 Pro Pro Leu Ala Ala Ile Glu Arg 20 15922PRTYersinia enterocolitica 159Asn Asn Tyr Ala Ile Thr His His Gly Tyr Tyr Asp Phe Gly Asn Ser 1 5 10 15 Thr Ser Tyr Val Gln Arg 20 16029PRTYersinia enterocolitica 160Ala Tyr Thr Asp Ile Thr Asp Ala Leu Lys Asp Val Pro Gly Val Val 1 5 10 15 Val Thr Gly Gly Gly Ser His Ser Asp Ile Ser Ile Arg 20 25 16127PRTYersinia enterocolitica 161Asn Gly Ala Ala Thr Phe Thr Leu Thr Pro Asp Asp Lys Asn Glu Phe 1 5 10 15 Asp Phe Asp Ile Gly His Tyr Val Gln Asp Arg 20 25 16211PRTYersinia enterocolitica 162Val Asn Phe Thr Ala Gly Val Gly Gly Tyr Arg 1 5 10 16312PRTYersinia enterocolitica 163Ser Ser Gln Ala Leu Ala Ile Gly Ser Gly Tyr Arg 1 5 10 16412PRTYersinia enterocolitica 164Asn Ser Val Ser Ile Gly His Glu Ser Leu Asn Arg 1 5 10 16515PRTYersinia enterocolitica 165Ala Ser Thr Ser Asp Thr Gly Val Ala Val Gly Phe Asn Ser Lys 1 5 10 15 16613PRTYersinia enterocolitica 166Thr Thr Leu Glu Thr Ala Glu Glu His Thr Asn Lys Lys 1 5 10 16715PRTYersinia enterocolitica 167Ser Ala Glu Thr Leu Ala Ser Ala Asn Val Tyr Ala Asp Ser Lys 1 5 10 15 16815PRTYersinia enterocolitica 168Ser Ala Glu Val Leu Gly Ile Ala Asn Asn Tyr Thr Asp Ser Lys 1 5 10 15 16917PRTYersinia enterocolitica 169Ala Leu Gly Asp Ser Ala Val Thr Tyr Gly Ala Gly Ser Thr Ala Gln 1 5 10 15 Lys 1707PRTYersinia enterocolitica 170Leu Gly Phe Ala Gly Leu Lys 1 5 1719PRTYersinia enterocolitica 171Ala Asp Ala Tyr Ser Gly Gly Leu Lys 1 5 1728PRTYersinia enterocolitica 172Asp Gly Asp Gln Ser Tyr Met Arg 1 5 17310PRTYersinia enterocolitica 173Asp Gly Asn Lys Leu Asp Leu Tyr Gly Lys 1 5 10 17411PRTYersinia enterocolitica 174Ala Glu Asp Gln Asp Gln Gly Asn Phe Thr Arg 1 5 10 17511PRTYersinia enterocolitica 175Val Asp Gly Leu His Tyr Phe Ser Asp Asp Lys 1 5 10 17611PRTYersinia enterocolitica 176Ile Asn Leu Leu Asp Glu Asn Glu Phe Thr Lys 1 5 10 17713PRTYersinia enterocolitica 177Val Asp Gly Leu His Tyr Phe Ser Asp Asp Lys Ser Lys 1 5 10 17818PRTYersinia enterocolitica 178Asn Ala Gly Ile Asn Thr Asp Asp Ile Val Ala Val Gly Leu Val Tyr 1 5 10 15 Gln Phe 17920PRTYersinia enterocolitica 179Asn Thr Asn Phe Phe Gly Leu Val Asp Gly Leu Asn Phe Ala Leu Gln 1 5 10 15 Tyr Gln Gly Lys 20 18020PRTYersinia enterocolitica 180Tyr Asp Ala Asn Asn Val Tyr Leu Ala Ala Thr Tyr Ala Gln Thr Tyr 1 5 10 15 Asn Leu Thr Arg 20 18123PRTYersinia enterocolitica 181Gly Glu Thr Gln Ile Ser Asp Gln Leu Thr Gly Tyr Gly Gln Trp Glu 1 5 10 15 Tyr Gln Ala Asn Leu Asn Lys 20 18226PRTYersinia enterocolitica 182Ala Gln Asn Ile Glu Leu Val Ala Gln Tyr Gln Phe Asp Phe Gly Leu 1 5 10 15 Arg Pro Ser Val Ala Tyr Leu Gln Ser Lys 20 25 18327PRTYersinia enterocolitica 183Phe Gly Leu Lys Gly Glu Thr Gln Ile Ser Asp Gln Leu Thr Gly Tyr 1 5 10 15 Gly Gln Trp Glu Tyr Gln Ala Asn Leu Asn Lys 20 25 1847PRTYersinia enterocolitica 184Thr Val Tyr Leu Gln Ile Lys 1 5 18513PRTYersinia enterocoliticaMOD_RES(7)..(7)Oxidized Met 185Asn Thr Ser Asp Lys Asn Met Leu Gly Leu Ala Pro Lys 1 5 10 18614PRTYersinia enterocolitica 186Phe Glu Glu Ala Gln Pro Val Leu Glu Asp Gln Leu Ala Lys 1 5 10 18715PRTYersinia enterocoliticaMOD_RES(3)..(3)Oxidized Met 187Thr Gln Met Ser Glu Thr Ile Trp Leu Glu Pro Ser Ser Gln Lys 1 5 10 15 18816PRTYersinia enterocolitica 188Val Gln Thr Ser Thr Gln Thr Gly Asn Lys His Gln Tyr Gln Thr Arg 1 5 10 15 18918PRTYersinia enterocolitica 189Val Asn Leu Lys Phe Glu Glu Ala Gln Pro Val Leu Glu Asp Gln Leu 1 5 10 15 Ala Lys 19021PRTYersinia enterocolitica 190Gly Tyr Thr Val Thr Ser Ser Pro Glu Asp Ala His Tyr Trp Ile Gln 1 5 10 15 Ala Asn Val Leu Lys 20 1916PRTYersinia pestis 191Ala Leu Ile Ser Leu Lys 1 5 1925PRTYersinia pestis 192Ser Ile Tyr Phe Arg 1 5 1937PRTYersinia pestis 193Ile Leu Ile Gly Glu Val Lys 1 5 1947PRTYersinia pestis 194Asn Pro Val Ala Arg Glu Arg 1 5 1958PRTYersinia pestis 195Ala Val Gln Asp Ile Ile Leu Lys 1 5 1968PRTYersinia pestis 196Tyr Pro Leu Ile Ser Glu Leu Lys 1 5 19710PRTYersinia pestis 197Asn

Gly Ile Ile Phe Ser Pro His Pro Arg 1 5 10 19812PRTYersinia pestis 198Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Lys 1 5 10 19911PRTYersinia pestis 199Asn Phe Glu Glu Ala Val Glu Lys Ala Glu Lys 1 5 10 20012PRTYersinia pestis 200Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys 1 5 10 20115PRTYersinia pestis 201Asn Gly Gly Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Thr Lys 1 5 10 15 20215PRTYersinia pestis 202Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro Leu Ala Lys 1 5 10 15 20314PRTYersinia pestis 203Ala Val Thr Asn Val Ala Glu Leu Asn Glu Leu Val Ala Arg 1 5 10 20413PRTYersinia pestis 204Gln Thr Ala Phe Ser Gln Tyr Asp Arg Pro Gln Ala Arg 1 5 10 20515PRTYersinia pestis 205Leu Leu Lys Glu Phe Leu Pro Ala Ser Tyr Asn Glu Gly Ala Lys 1 5 10 15 20616PRTYersinia pestis 206Tyr Ala Glu Ile Ala Asp His Leu Gly Leu Ser Ala Pro Gly Asp Arg 1 5 10 15 20717PRTYersinia pestis 207Gly Ser Leu Pro Ile Ala Leu Glu Glu Val Ala Thr Asp Gly Ala Lys 1 5 10 15 Arg 20815PRTYersinia pestis 208Glu Tyr Ala Asn Phe Ser Gln Glu Gln Val Asp Lys Ile Phe Arg 1 5 10 15 20916PRTYersinia pestis 209Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr Lys Asp Glu Lys 1 5 10 15 21019PRTYersinia pestis 210Ile Leu Ile Asn Thr Pro Ala Ser Gln Gly Gly Ile Gly Asp Leu Tyr 1 5 10 15 Asn Phe Lys 21119PRTYersinia pestis 211Glu Tyr Val Glu Glu Phe Asp Arg Glu Glu Glu Val Ala Ala Ala Thr 1 5 10 15 Ala Pro Lys 21222PRTYersinia pestis 212Tyr Asn Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr 1 5 10 15 Asp Arg Pro Gln Ala Arg 20 21329PRTYersinia pestis 213Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile Gly Val Gly Ala Gly Asn 1 5 10 15 Thr Pro Val Val Val Asp Glu Thr Ala Asp Ile Lys Arg 20 25 21410PRTYersinia pestis 214Ile Leu Phe Tyr Thr Gly Val Asn His Lys 1 5 10 21514PRTYersinia pestis 215Tyr Arg Asn Ile Gly Ile Ser Ala His Ile Asp Ala Gly Lys 1 5 10 21614PRTYersinia pestis 216His Ser Asp Asp Lys Glu Pro Phe Ser Ala Leu Ala Phe Lys 1 5 10 21714PRTYersinia pestis 217Ile Ala Thr Asp Pro Phe Val Gly Asn Leu Thr Phe Phe Arg 1 5 10 21814PRTYersinia pestis 218Tyr Leu Gly Gly Glu Glu Leu Thr Glu Glu Glu Ile Lys Lys 1 5 10 21915PRTYersinia pestis 219Met Glu Phe Pro Glu Pro Val Ile Ser Val Ala Val Glu Pro Lys 1 5 10 15 22015PRTYersinia pestis 220Glu Phe Ile Pro Ala Val Asp Lys Gly Ile Gln Glu Gln Leu Lys 1 5 10 15 22117PRTYersinia pestis 221Leu Gly Ala Asn Pro Val Pro Leu Gln Leu Ala Ile Gly Ala Glu Glu 1 5 10 15 Lys 22217PRTYersinia pestis 222Val Tyr Ser Gly Ile Val Asn Ser Gly Asp Thr Val Leu Asn Ser Val 1 5 10 15 Lys 22316PRTYersinia pestis 223Glu Phe Asn Val Glu Ala Asn Val Gly Lys Pro Gln Val Ala Tyr Arg 1 5 10 15 22418PRTYersinia pestis 224Glu Glu Ile Lys Glu Val His Ala Gly Asp Ile Ala Ala Ala Ile Gly 1 5 10 15 Leu Lys 22517PRTYersinia pestis 225Leu His Tyr Gly Ser Tyr His Asp Val Asp Ser Ser Glu Leu Ala Phe 1 5 10 15 Lys 22620PRTYersinia pestis 226Val Tyr Ser Gly Ile Val Asn Ser Gly Asp Thr Val Leu Asn Ser Val 1 5 10 15 Lys Ser Gln Arg 20 2277PRTYersinia pestis 227Asn Arg Asp Glu Trp Ser Arg 1 5 2289PRTYersinia pestisMOD_RES(5)..(5)Oxidized Met 228Tyr Glu Tyr Gly Met Phe Ser Gln Lys 1 5 22911PRTYersinia pestis 229Val Ser Val Ile Asp Glu Asn Asn Gly Arg Arg 1 5 10 23012PRTYersinia pestis 230Val Leu Tyr Pro Asp Asp Ser Thr Tyr Ser Gly Arg 1 5 10 23114PRTYersinia pestis 231Glu Glu Asn Asp Pro Gly Leu Gly Asn Gly Gly Leu Gly Arg 1 5 10 23212PRTYersinia pestis 232Ile Ile Asp Ala Pro Asp Asn Asn Trp Val Pro Arg 1 5 10 23313PRTYersinia pestis 233Asn Leu Asp Tyr Pro Ser Phe Leu Leu Ala Leu Gln Lys 1 5 10 23413PRTYersinia pestis 234Glu Tyr Ala Asp Glu Ile Trp His Ile Lys Pro Ile Arg 1 5 10 23514PRTYersinia pestis 235Ser Tyr Val Asp Thr Gln Glu Gln Val Asp Ala Leu Tyr Arg 1 5 10 23614PRTYersinia pestisMOD_RES(10)..(10)Oxidized Met 236Gly Tyr Gly Ile Arg Tyr Glu Tyr Gly Met Phe Ser Gln Lys 1 5 10 23715PRTYersinia pestisMOD_RES(8)..(8)Oxidized Met 237Thr Leu Leu Asn Ile Ala Asn Met Gly Tyr Phe Ser Ser Asp Arg 1 5 10 15 23817PRTYersinia pestis 238Thr Ser Pro Phe Ser Tyr Thr Ser Pro Val Val Ser Val Asp Ala Leu 1 5 10 15 Lys 23915PRTYersinia pestis 239Leu Val Glu Glu Gln Tyr Pro Asp Asp Lys Glu Leu Leu Ser Arg 1 5 10 15 24016PRTYersinia pestisMOD_RES(9)..(9)Oxidized Met 240Lys Thr Leu Leu Asn Ile Ala Asn Met Gly Tyr Phe Ser Ser Asp Arg 1 5 10 15 24119PRTYersinia pestisMOD_RES(17)..(18)Oxidized Met 241Ile Ala Ile His Leu Asn Asp Thr His Pro Val Leu Ser Ile Pro Glu 1 5 10 15 Met Met Arg 24221PRTYersinia pestis 242Phe Asn Gln Gly Asp Tyr Phe Ala Ala Val Glu Asp Lys Asn His Ser 1 5 10 15 Glu Asn Val Ser Arg 20 2438PRTYersinia pestis 243Tyr Ile Gln Ala Ala Val Pro Lys 1 5 2447PRTYersinia pestis 244Phe Asn Ile Asn Tyr Thr Arg 1 5 2459PRTYersinia pestis 245Ser Gly Phe Leu Ile Pro Asn Ala Lys 1 5 2468PRTYersinia pestis 246Ile Gly Phe Asn Ile Glu Leu Arg 1 5 2479PRTYersinia pestis 247Ala Gln Tyr Leu Tyr Val Pro Tyr Arg 1 5 2489PRTYersinia pestis 248Gly Leu Gln Trp Gln Asn Glu Phe Arg 1 5 24912PRTYersinia pestis 249Ile Thr Gly Trp Asn Ala Gln Gly Gln Thr Ser Lys 1 5 10 25010PRTYersinia pestis 250Arg Gly Leu Gln Trp Gln Asn Glu Phe Arg 1 5 10 25111PRTYersinia pestis 251Glu Glu Gln Val Val Glu Val Trp Asn Ala Arg 1 5 10 25214PRTYersinia pestis 252Ile Ala Ser Ala Asn Gln Val Ser Thr Gly Leu Thr Ser Arg 1 5 10 25313PRTYersinia pestis 253Phe Thr Ser Val Asn Pro Thr Asn Pro Glu Ala Ser Arg 1 5 10 25414PRTYersinia pestis 254Ile Tyr Thr Gly Pro Asp Gly Thr Asp Lys Asn Ala Thr Arg 1 5 10 25513PRTYersinia pestis 255Phe Asn Val Ser Val Gly Gln Ile Tyr Tyr Phe Ser Arg 1 5 10 25615PRTYersinia pestis 256Gln Phe Gln Val Phe Thr Ala Ala Gly Asn Ser Asn Ala Tyr Arg 1 5 10 15 25716PRTYersinia pestis 257Thr Val Thr Ala Thr Gly Asp Val Asn Tyr Asp Asp Pro Gln Ile Lys 1 5 10 15 25822PRTYersinia pestis 258Leu Leu Ala Thr His Tyr Gln Gln Asp Ile Pro Ala Ser Phe Ala Asp 1 5 10 15 Asn Ala Ser Asn Pro Lys 20 25924PRTYersinia pestis 259Val Tyr Asn Pro Asp Tyr Gln Gln Gly Ile Ser Gln Val Gly Thr Thr 1 5 10 15 Ala Ser Trp Pro Ile Ala Asp Arg 20 2606PRTYersinia pestis 260Asp Ile Gly Asn Ile Arg 1 5 2616PRTYersinia pestis 261Arg Ile Glu Ile Val Arg 1 5 2627PRTYersinia pestis 262Val Ser Tyr Phe Asp Thr Lys 1 5 2638PRTYersinia pestis 263Ala Lys Asp Tyr Ile Ser Thr Arg 1 5 26411PRTYersinia pestis 264Asp Leu Pro Val Ser Ile Leu Ala Gly Thr Arg 1 5 10 26511PRTYersinia pestis 265Gln Gly Val Leu Thr Leu Val Asp Gly Val Arg 1 5 10 26612PRTYersinia pestis 266Gln Val Pro Gly Leu Thr Val Thr Gly Ser Gly Arg 1 5 10 26710PRTYersinia pestis 267Tyr Tyr Asn Asn Ser Ala Ile Glu Pro Lys 1 5 10 26812PRTYersinia pestis 268Glu Gln Thr Thr Glu Gly Val Lys Leu Glu Asn Arg 1 5 10 26913PRTYersinia pestis 269Thr Asp Asp Leu Asp Gly Ile Leu Ser Phe Gly Thr Arg 1 5 10 27012PRTYersinia pestis 270Thr Ala Leu Phe Asn Trp Asp Leu Ala Tyr Asn Arg 1 5 10 27113PRTYersinia pestis 271Glu Tyr Tyr Thr Pro Gln Gly Ile Pro Gln Asp Gly Arg 1 5 10 27213PRTYersinia pestis 272Phe Ser Ser Gly Trp Leu Gln Asp Glu Ile Thr Leu Arg 1 5 10 27315PRTYersinia pestis 273His Ser Thr Asp Thr Met Val Val Thr Ala Thr Gly Asn Glu Arg 1 5 10 15 27416PRTYersinia pestis 274Gln Glu Gln Thr Pro Gly Gly Ala Thr Glu Ser Phe Pro Gln Ala Lys 1 5 10 15 27516PRTYersinia pestis 275Lys His Ser Thr Asp Thr Met Val Val Thr Ala Thr Gly Asn Glu Arg 1 5 10 15 27616PRTYersinia pestis 276Gly Thr Trp Gln Ile Asp Ser Ile Gln Ser Leu Ser Ala Asn Leu Arg 1 5 10 15 27715PRTYersinia pestis 277Ile Arg Phe Ser Ser Gly Trp Leu Gln Asp Glu Ile Thr Leu Arg 1 5 10 15 27817PRTYersinia pestis 278Val Asp Met Gln Ala Met Thr Thr Thr Ser Val Asn Ile Asp Gln Ala 1 5 10 15 Lys 27918PRTYersinia pestis 279Tyr Asp Asn Tyr Ser Gly Ser Ser Asp Gly Tyr Ala Asp Val Asp Ala 1 5 10 15 Asp Lys 28019PRTYersinia pestis 280Gln Gly Thr Asp Thr Gly His Leu Asn Ser Thr Phe Leu Asp Pro Ala 1 5 10 15 Leu Val Lys 28119PRTYersinia pestis 281Gln Ser Asn Gly Phe Asn Ala Pro Asn Asp Glu Thr Ile Ser Asn Val 1 5 10 15 Leu Ala Lys 28221PRTYersinia pestis 282Val Tyr Ser Ser Ala Ala Thr Gly Asp His Ser Phe Gly Leu Gly Ala 1 5 10 15 Ser Ala Phe Gly Arg 20 28320PRTYersinia pestis 283Val Ser Ser Ser Thr Pro Gln Ala Gly Tyr Gly Val Asn Asp Phe Tyr 1 5 10 15 Val Ser Tyr Lys 20 28419PRTYersinia pestis 284Leu Phe Ile Glu Ser Pro Ala Ser His Leu Leu Thr Tyr Gly Thr Glu 1 5 10 15 Thr Tyr Lys 28521PRTYersinia pestis 285Thr Arg Leu Phe Ile Glu Ser Pro Ala Ser His Leu Leu Thr Tyr Gly 1 5 10 15 Thr Glu Thr Tyr Lys 20 28622PRTYersinia pestis 286Tyr Asp Asn Tyr Ser Gly Ser Ser Asp Gly Tyr Ala Asp Val Asp Ala 1 5 10 15 Asp Lys Trp Ser Ser Arg 20 28726PRTYersinia pestis 287Val Ser Ser Ser Thr Pro Gln Ala Gly Tyr Gly Val Asn Asp Phe Tyr 1 5 10 15 Val Ser Tyr Lys Gly Gln Glu Ala Phe Lys 20 25 2885PRTYersinia pestis 288Ile Glu Val Ile Arg 1 5 2896PRTYersinia pestis 289Gly Thr Ile Phe Arg Arg 1 5 2908PRTYersinia pestis 290Gly Gly Tyr Glu Asp Thr Leu Arg 1 5 2919PRTYersinia pestis 291Thr Gly Gly Leu Asp Ile Ser Ile Arg 1 5 29212PRTYersinia pestis 292Leu Leu Asp Ser Leu Ala Leu Thr Tyr Gly Ala Arg 1 5 10 29312PRTYersinia pestis 293Leu Leu Lys Asn Thr Asn Ile Ile Leu Asp Ser Lys 1 5 10 29413PRTYersinia pestis 294Phe Thr Gln Asn Tyr Ala Asn Leu Ser Ala Ala Asn Lys 1 5 10 29514PRTYersinia pestis 295Tyr Asp Asn Ser Ala Asn Gln Leu Gly Thr Ile Gly Ala Arg 1 5 10 29615PRTYersinia pestis 296Glu Ala Ala Ala Ser Ile Ser Val Ile Ser Gln Asn Glu Leu Arg 1 5 10 15 29715PRTYersinia pestis 297Gly Met Pro Ser Ala Tyr Thr Leu Ile Leu Val Asp Gly Ile Arg 1 5 10 15 29817PRTYersinia pestis 298Leu Ile Thr Asn Ala Ser Val Pro Gln Gly Ser Gly Leu Ala Gly Glu 1 5 10 15 Lys 29914PRTYersinia pestis 299Tyr Glu Tyr Gln Thr Thr Phe Gly Gly His Ile Ser Pro Arg 1 5 10 30016PRTYersinia pestis 300Asp Ala Ser Arg Val Glu Ser Ser Asn Thr Gly Val Glu Leu Ser Arg 1 5 10 15 30114PRTYersinia pestis 301Ala Tyr Leu Val Trp Asp Ala Gln Asp Asn Trp Thr Val Lys 1 5 10 30214PRTYersinia pestis 302Leu Asn Trp Asn Ile Asn Glu Gln Leu Ser Thr Trp Leu Lys 1 5 10 30318PRTYersinia pestis 303Leu Ile Thr Asn Ala Ser Val Pro Gln Gly Ser Gly Leu Ala Gly Glu 1 5 10 15 Lys Arg 30418PRTYersinia pestis 304Ile Asn Ser Val Ser Ile Asp Asn Thr Thr Ser Thr Tyr Thr Asn Val 1 5 10 15 Gly Lys 30518PRTYersinia pestis 305Asp Val Thr Leu Asn Gly Ala Val Asn Asn Leu Leu Asp Lys Asp Phe 1 5 10 15 Thr Arg 30619PRTYersinia pestis 306Phe Ser Phe Tyr Ser Ser Gly Pro Ala Val Glu Asp Gln Leu Gly Leu 1 5 10 15 Ser Leu Arg 30720PRTYersinia pestis 307Asn Lys Ile Asn Ser Val Ser Ile Asp Asn Thr Thr Ser Thr Tyr Thr 1 5 10 15 Asn Val Gly Lys 20 30820PRTYersinia pestis 308Leu Asp Phe Gly Thr Trp Asn Ser Ser Leu Ser Tyr Asn Gln Thr Glu 1 5 10 15 Asn Ile Gly Arg 20 30923PRTYersinia pestis 309Asn Tyr Asn Asp Leu Ala Gln Ala Leu Ser Asp Val Glu Gly Val Asp 1 5 10 15 Val Asn Ser Ser Thr Gly Lys 20 31023PRTYersinia pestis 310Ala Trp Ala Ser Ser Ala Thr Leu Glu His Thr Phe Gln Glu Asn Thr 1 5 10 15 Ala Phe Gly Asp Ser Ser Lys 20 31123PRTYersinia pestis 311Val Val Tyr Asn Asn Leu Gly Ser Glu Phe Lys Pro Phe Ser Val Leu 1 5 10 15 Asn Leu Gly Val Ala Tyr Lys 20 31223PRTYersinia pestis 312Val Val Tyr Asn Asn Leu Gly Ser Glu Phe Lys Pro Phe Ser Val Leu 1 5 10 15 Asn Leu Gly Val Ala Tyr Lys 20 31327PRTYersinia pestis 313Thr Pro Thr Leu Ala Gln Leu His Asn Gly Ile Ser Gly Val Thr Gly 1 5 10 15 Gln Gly Thr Ile Thr Thr Ile Gly Asn Pro Lys 20 25 31426PRTYersinia pestis 314Asp Gly Ile Val Leu Ala Asn Asn Gly Asp Glu Phe Ala Gln Asp Ala 1 5 10 15 Trp Ser Leu Phe Ser Glu Asp Glu Trp Arg 20 25 31531PRTYersinia pestis 315Thr His Ile Phe Ala Val Gly Asn Gly Thr Thr Thr Ala Gly Asp Tyr 1 5 10 15 Phe Thr Ser Ser Gln Ser Thr Ala Gly Tyr Val Val Pro Gly Arg 20 25 30 31628PRTYersinia pestis 316Ile Thr Leu Gly Asn Asp Asn Arg Leu Asp Phe Gly Thr Trp Asn Ser 1 5 10 15 Ser Leu Ser Tyr Asn Gln Thr Glu Asn Ile Gly Arg 20 25 31735PRTYersinia pestis 317Gly Gly Val Ser Thr Gly Tyr Lys Thr Pro Thr Leu Ala Gln Leu His 1 5 10 15 Asn Gly Ile Ser Gly Val Thr Gly Gln Gly Thr Ile Thr Thr Ile Gly 20

25 30 Asn Pro Lys 35 31831PRTYersinia pestis 318Leu Glu Pro Glu Ser Ser Val Asn Thr Glu Val Gly Val Tyr Tyr Glu 1 5 10 15 Asn Glu Thr Gly Phe Gly Ala Asn Val Thr Leu Phe His Asn Arg 20 25 30 3196PRTYersinia pestis 319Val Pro Phe Val Pro Arg 1 5 3207PRTYersinia pestis 320Thr Val Gly Ile Asn Thr Arg 1 5 3218PRTYersinia pestis 321Ala Ala Thr Leu Gly Asp Ala Arg 1 5 3227PRTYersinia pestis 322Tyr Gly Ala Leu Met Pro Arg 1 5 3239PRTYersinia pestis 323Gly Pro Gln Gly Thr Leu Tyr Gly Lys 1 5 32410PRTYersinia pestis 324Gly Tyr Ile Glu Gly Gly Val Ser Ser Arg 1 5 10 3259PRTYersinia pestis 325Ser Ile Asn Tyr Glu Leu Gly Thr Arg 1 5 32611PRTYersinia pestis 326Ala Asp Ala Thr Gly Val Glu Leu Glu Ala Lys 1 5 10 32710PRTYersinia pestis 327Asp Met Gln Leu Tyr Ser Gly Pro Val Arg 1 5 10 3289PRTYersinia pestis 328Trp Asn Gln Asp Val Gln Glu Leu Arg 1 5 32910PRTYersinia pestis 329Thr Val Asp Met Val Phe Gly Leu Tyr Arg 1 5 10 33011PRTYersinia pestis 330Thr Val Gly Ile Asn Thr Arg Ile Asp Phe Phe 1 5 10 33114PRTYersinia pestis 331Tyr Gly Ala Gly Ser Ser Val Asn Gly Val Ile Asp Thr Arg 1 5 10 33213PRTYersinia pestis 332Ala Asp Ala Thr Gly Val Glu Leu Glu Ala Lys Trp Arg 1 5 10 33313PRTYersinia pestis 333Ala Thr Gln Asp Ala Tyr Val Gly Trp Asn Asp Ile Lys 1 5 10 33414PRTYersinia pestis 334Thr Phe Pro Ser Gly Ser Leu Ile Val Asn Met Pro Gln Arg 1 5 10 33514PRTYersinia pestis 335Ser Glu Phe Thr Asn Asp Ser Glu Leu Tyr His Gly Asn Arg 1 5 10 33615PRTYersinia pestis 336Ala Thr Gln Asp Ala Tyr Val Gly Trp Asn Asp Ile Lys Gly Arg 1 5 10 15 33715PRTYersinia pestis 337Phe Ala Pro Gly Trp Ser Trp Asp Ile Asn Gly Asn Val Ile Arg 1 5 10 15 33816PRTYersinia pestis 338Leu Ala Pro Asp Asp Gln Pro Trp Glu Met Gly Phe Ala Ala Ser Arg 1 5 10 15 33918PRTYersinia pestis 339Thr Tyr Gly Tyr Met Asn Gly Ser Ser Ala Val Ala Gln Val Asn Met 1 5 10 15 Gly Arg 34017PRTYersinia pestis 340Glu Cys Thr Arg Ala Thr Gln Asp Ala Tyr Val Gly Trp Asn Asp Ile 1 5 10 15 Lys 34119PRTYersinia pestis 341Ser Ala Gln Gly Gly Ile Ile Asn Ile Val Thr Gln Gln Pro Asp Ser 1 5 10 15 Thr Pro Arg 34219PRTYersinia pestis 342Ser Ser Thr Gln Tyr His Gly Ser Met Leu Gly Asn Pro Phe Gly Asp 1 5 10 15 Gln Gly Lys 34318PRTYersinia pestis 343Leu Ala Val Asn Leu Val Gly Pro His Tyr Phe Asp Gly Asp Asn Gln 1 5 10 15 Leu Arg 34418PRTYersinia pestis 344Tyr Glu Thr Ala Asp Val Thr Leu Gln Ala Ala Thr Phe Tyr Thr His 1 5 10 15 Thr Lys 34520PRTYersinia pestis 345Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly Ser Val 1 5 10 15 Thr Leu Leu Arg 20 34620PRTYersinia pestis 346Ser Glu Phe Thr Asn Asp Ser Glu Leu Tyr His Gly Asn Arg Val Pro 1 5 10 15 Phe Val Pro Arg 20 34722PRTYersinia pestis 347Ser Lys Phe Asn Leu Ser Gly Pro Ile Gln Asp Gly Leu Leu Tyr Gly 1 5 10 15 Ser Val Thr Leu Leu Arg 20 34827PRTYersinia pestis 348Val Ala Gln Gly Tyr Lys Pro Ser Gly Tyr Asn Ile Val Pro Thr Ala 1 5 10 15 Gly Leu Asp Ala Lys Pro Phe Val Ala Glu Lys 20 25 34930PRTYersinia pestis 349Ser Ala Ser Ala Asn Asn Val Ser Ser Thr Val Val Ser Ala Pro Glu 1 5 10 15 Leu Ser Asp Ala Gly Val Thr Ala Ser Asp Lys Leu Pro Arg 20 25 30 3509PRTYersinia pestis 350Val Glu Asp Ala Leu His Ala Thr Arg 1 5 35112PRTYersinia pestis 351Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg 1 5 10 35211PRTYersinia pestis 352Thr Thr Leu Glu Asp Leu Gly Gln Ala Lys Arg 1 5 10 35311PRTYersinia pestis 353Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg 1 5 10 35412PRTYersinia pestis 354Val Gly Ala Ala Thr Glu Val Glu Met Lys Glu Lys 1 5 10 35517PRTYersinia pestis 355Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly Val Ala Leu Ile 1 5 10 15 Arg 35615PRTYersinia pestis 356Asn Val Val Leu Asp Lys Ser Phe Gly Ser Pro Thr Ile Thr Lys 1 5 10 15 35716PRTYersinia pestis 357Ser Phe Gly Ser Pro Thr Ile Thr Lys Asp Gly Val Ser Val Ala Arg 1 5 10 15 35814PRTYersinia pestis 358Gln Gln Ile Glu Asp Ala Thr Ser Asp Tyr Asp Lys Glu Lys 1 5 10 35920PRTYersinia pestis 359Ala Ala His Ala Ile Ala Gly Leu Lys Gly Asp Asn Glu Asp Gln Asn 1 5 10 15 Val Gly Ile Lys 20 36023PRTYersinia pestis 360Val Val Ile Asn Lys Asp Thr Thr Ile Ile Ile Asp Gly Val Gly Asp 1 5 10 15 Glu Ala Ala Ile Gln Gly Arg 20 3618PRTYersinia pestis 361Asn Leu Ser Leu Leu Ser Ala Arg 1 5 3629PRTYersinia pestis 362Gln Thr Val Thr Thr Pro Arg Ala Gln 1 5 36311PRTYersinia pestis 363Ala Ala Ala Asp Arg Asp Ala Ala Tyr Glu Lys 1 5 10 36411PRTYersinia pestis 364Asn Asn Leu Asp Asn Ala Leu Glu Ser Leu Arg 1 5 10 36511PRTYersinia pestis 365Leu Ser Gln Asp Leu Ala Arg Glu Gln Ile Lys 1 5 10 36611PRTYersinia pestis 366Asp Ala Ala Tyr Glu Lys Ile Asn Glu Val Arg 1 5 10 36712PRTYersinia pestis 367Ala Ile Asp Ser Leu Ser Tyr Thr Glu Ala Gln Lys 1 5 10 36812PRTYersinia pestis 368Thr Gln Arg Pro Asp Ala Val Asn Asn Leu Leu Lys 1 5 10 36911PRTYersinia pestis 369Tyr Asn Tyr Leu Ile Asn Gln Leu Asn Ile Lys 1 5 10 37014PRTYersinia pestis 370Ala Ser Tyr Asp Thr Val Leu Ala Ala Glu Val Ala Ala Arg 1 5 10 37114PRTYersinia pestis 371Leu Lys Thr Gln Arg Pro Asp Ala Val Asn Asn Leu Leu Lys 1 5 10 37215PRTYersinia pestis 372Phe Asn Val Gly Leu Val Ala Ile Thr Asp Val Gln Asn Ala Arg 1 5 10 15 37316PRTYersinia pestis 373Thr Ile Leu Asp Val Leu Thr Ala Thr Thr Asn Leu Tyr Gln Ser Lys 1 5 10 15 37417PRTYersinia pestis 374Gln Ile Thr Gly Val Tyr Tyr Pro Glu Leu Ala Ser Leu Asn Val Glu 1 5 10 15 Arg 37517PRTYersinia pestis 375Ala Ile Asp Ser Leu Ser Tyr Thr Glu Ala Gln Lys Gln Ser Val Tyr 1 5 10 15 Arg 37618PRTYersinia pestis 376Gln Ala Gln Tyr Asn Phe Val Gly Ala Ser Glu Leu Leu Glu Ser Ala 1 5 10 15 His Arg 37720PRTYersinia pestis 377Ser Pro Leu Leu Pro Gln Leu Gly Leu Ser Ala Gly Tyr Thr His Ala 1 5 10 15 Asn Gly Phe Arg 20 37818PRTYersinia pestis 378Gln Gln Leu Ala Asp Ala Arg Tyr Asn Tyr Leu Ile Asn Gln Leu Asn 1 5 10 15 Ile Lys 37925PRTYersinia pestis 379Ile Asn Glu Val Arg Ser Pro Leu Leu Pro Gln Leu Gly Leu Ser Ala 1 5 10 15 Gly Tyr Thr His Ala Asn Gly Phe Arg 20 25 3806PRTYersinia pestis 380His Thr Pro Phe Phe Lys 1 5 3817PRTYersinia pestis 381Glu His Ile Leu Leu Gly Arg 1 5 3828PRTYersinia pestis 382Phe Ala Ile Arg Glu Gly Gly Arg 1 5 38310PRTYersinia pestis 383Ala Gly Glu Asn Val Gly Val Leu Leu Arg 1 5 10 38410PRTYersinia pestis 384Gly Thr Val Val Thr Gly Arg Val Glu Arg 1 5 10 38513PRTYersinia pestis 385Glu Gly Gly Arg Thr Val Gly Ala Gly Val Val Ala Lys 1 5 10 38611PRTYersinia pestis 386Ala Leu Glu Gly Glu Ala Glu Trp Glu Ala Lys 1 5 10 3879PRTYersinia pestis 387Gly Tyr Arg Pro Gln Phe Tyr Phe Arg 1 5 38811PRTYersinia pestis 388Asp Glu Gly Gly Arg His Thr Pro Phe Phe Lys 1 5 10 38912PRTYersinia pestis 389Ala Phe Asp Gln Ile Asp Asn Ala Pro Glu Glu Lys 1 5 10 39014PRTYersinia pestis 390Ala Phe Asp Gln Ile Asp Asn Ala Pro Glu Glu Lys Ala Arg 1 5 10 39115PRTYersinia pestis 391Val Gly Glu Glu Val Glu Ile Val Gly Ile Lys Asp Thr Val Lys 1 5 10 15 39216PRTYersinia pestis 392Leu Leu Asp Glu Gly Arg Ala Gly Glu Asn Val Gly Val Leu Leu Arg 1 5 10 15 39316PRTYersinia pestis 393Gly Ile Thr Ile Asn Thr Ser His Val Glu Tyr Asp Thr Pro Ala Arg 1 5 10 15 39417PRTYersinia pestis 394Thr Lys Pro His Val Asn Val Gly Thr Ile Gly His Val Asp His Gly 1 5 10 15 Lys 39517PRTYersinia pestis 395Glu Leu Leu Ser Ala Tyr Asp Phe Pro Gly Asp Asp Leu Pro Val Val 1 5 10 15 Arg 39617PRTYersinia pestis 396Ile Ile Glu Leu Ala Gly Tyr Leu Asp Ser Tyr Ile Pro Glu Pro Glu 1 5 10 15 Arg 39718PRTYersinia pestis 397Ala Arg Gly Ile Thr Ile Asn Thr Ser His Val Glu Tyr Asp Thr Pro 1 5 10 15 Ala Arg 3987PRTYersinia pestis 398Val Gly Phe Ala Gly Leu Lys 1 5 3999PRTYersinia pestis 399Ala Asn Ala Tyr Thr Gly Gly Leu Lys 1 5 4008PRTYersinia pestis 400Gly Asn Gly Met Leu Thr Tyr Arg 1 5 40110PRTYersinia pestis 401Arg Ala Asn Ala Tyr Thr Gly Gly Leu Lys 1 5 10 40211PRTYersinia pestis 402Ser Ser Asp Ala Ala Phe Gly Phe Ala Asp Lys 1 5 10 4039PRTYersinia pestis 403Asn Met Ser Thr Tyr Val Asp Tyr Lys 1 5 40411PRTYersinia pestis 404Asn Gly Ser Ser Ser Glu Thr Asn Asn Gly Arg 1 5 10 40510PRTYersinia pestis 405Asn Leu Asp Gly Asp Gln Ser Tyr Met Arg 1 5 10 40611PRTYersinia pestis 406Phe Ala Asp Tyr Gly Ser Leu Asp Tyr Gly Arg 1 5 10 40711PRTYersinia pestis 407Ile Asp Gly Leu His Tyr Phe Ser Asp Asn Lys 1 5 10 40811PRTYersinia pestis 408Ile Asn Leu Leu Asp Lys Asn Asp Phe Thr Lys 1 5 10 40913PRTYersinia pestis 409Thr Thr Ala Gln Asn Asp Leu Gln Tyr Gly Gln Gly Lys 1 5 10 41012PRTYersinia pestis 410Tyr Val Asp Ile Gly Ala Thr Tyr Phe Phe Asn Lys 1 5 10 41113PRTYersinia pestis 411Ala Glu Asn Glu Asp Gly Asn His Asp Ser Phe Thr Arg 1 5 10 41214PRTYersinia pestis 412Gly Lys Asp Ile Gly Ile Tyr Gly Asp Gln Asp Leu Leu Lys 1 5 10 41314PRTYersinia pestis 413Thr Thr Ala Gln Asn Asp Leu Gln Tyr Gly Gln Gly Lys Arg 1 5 10 41420PRTYersinia pestis 414Asn Thr Asn Phe Phe Gly Leu Val Asp Gly Leu Asn Phe Ala Leu Gln 1 5 10 15 Tyr Gln Gly Lys 20 41520PRTYersinia pestis 415Tyr Asp Ala Asn Asn Val Tyr Leu Ala Ala Asn Tyr Thr Gln Thr Tyr 1 5 10 15 Asn Leu Thr Arg 20 41621PRTYersinia pestis 416Ile Asp Gly Leu His Tyr Phe Ser Asp Asn Lys Asn Leu Asp Gly Asp 1 5 10 15 Gln Ser Tyr Met Arg 20 41723PRTYersinia pestis 417Gly Glu Thr Gln Ile Thr Asp Gln Leu Thr Gly Tyr Gly Gln Trp Glu 1 5 10 15 Tyr Gln Val Asn Leu Asn Lys 20 41826PRTYersinia pestis 418Ala His Asn Ile Glu Val Val Ala Gln Tyr Gln Phe Asp Phe Gly Leu 1 5 10 15 Arg Pro Ser Val Ala Tyr Leu Gln Ser Lys 20 25 41933PRTYersinia pestis 419Gly Val Ala Asp Gln Asn Gly Asp Gly Tyr Gly Met Ser Leu Ser Tyr 1 5 10 15 Asp Leu Gly Trp Gly Val Ser Ala Ser Ala Ala Met Ala Ser Ser Leu 20 25 30 Arg 4209PRTYersinia pestis 420Ala Leu Ala Ser Asn Ile Leu Tyr Arg 1 5 42110PRTYersinia pestis 421Ser Asp Pro Gly Ala Ala Phe Pro Trp Lys 1 5 10 42211PRTYersinia pestis 422Lys Ser Asp Pro Gly Ala Ala Phe Pro Trp Lys 1 5 10 42310PRTYersinia pestis 423Ile Phe Asn Leu Val Asp Glu Asn Glu Arg 1 5 10 42410PRTYersinia pestis 424Met Tyr Asn Ile Asp Tyr Asn Ser Phe Arg 1 5 10 42512PRTYersinia pestis 425Ala Trp His Ala Gly Val Ser Tyr Trp Asp Gly Arg 1 5 10 42616PRTYersinia pestis 426Ala Leu Tyr Asp Ala Gly Ile Gly Ala Trp Tyr Asp Asp Glu Thr Lys 1 5 10 15 42719PRTYersinia pestis 427Phe Pro Asp Ile Thr Pro Val Asn Val Val Gly His Ser Asp Ile Ala 1 5 10 15 Pro Gly Arg 42819PRTYersinia pestis 428Tyr Gly Tyr Asp Thr Ser Gly Ala Val Ser Glu Val Gly Tyr Asn Gln 1 5 10 15 Leu Ile Arg 42920PRTYersinia pestis 429Phe Pro Asp Ile Thr Pro Val Asn Val Val Gly His Ser Asp Ile Ala 1 5 10 15 Pro Gly Arg Lys 20 43010PRTYersinia pestis 430Ser Asp Pro Gly Pro Leu Phe Pro Trp Lys 1 5 10 43111PRTYersinia pestis 431Ser Asp Pro Gly Pro Leu Phe Pro Trp Lys Arg 1 5 10 43212PRTYersinia pestis 432Ala Ile Ala Leu Gln Leu Val Pro Glu Ala Gln Arg 1 5 10 43312PRTYersinia pestis 433Ala Trp His Ala Gly Val Ser Ser Trp Gln Gly Arg 1 5 10 43412PRTYersinia pestis 434Ile Pro Gln Asn Gly Gln Leu Asp Thr Glu Thr Arg 1 5 10 43514PRTYersinia pestis 435Gly Thr Tyr Gln Ile Asp Thr His Tyr Pro Ser Val Ala Lys 1 5 10 43618PRTYersinia pestis 436Gly Ala Ala Ser Val Ala Val Ile Gln Gln Ala Leu Ala Ala Tyr Gly 1 5 10 15 Tyr Lys 43716PRTYersinia pestis 437Phe Leu Val Leu His Tyr Thr Ala Val Gly Asp Ala Glu Ser Leu Arg 1 5 10 15 43818PRTYersinia pestis 438Tyr Asn Ile Ser Pro Ser Asp Val Val Ala His Ser Asp Ile Ala Pro 1 5 10 15 Leu Arg 43920PRTYersinia pestis 439Asn Asn Leu Asn Asp Thr Ser Ile Gly Ile Glu Ile Val Asn Leu Gly 1 5 10 15 Phe Thr Glu Lys 20 44024PRTYersinia pestis 440Ala Ile Ala Leu Gln Leu Val Pro Glu Ala Gln Arg Ala Trp His Ala 1 5 10 15 Gly Val Ser Ser Trp Gln Gly Arg 20 4417PRTYersinia pestis 441Leu Ile Asp Gly Asp Phe Lys 1 5 44210PRTYersinia pestis 442Gly Phe Glu Glu Ser Val Asp Gly Phe Lys 1 5 10 44311PRTYersinia pestis 443Val Gly Thr Trp Met Leu Gly Ala Gly Tyr Arg 1 5 10 44411PRTYersinia pestis 444Phe Ser Ser Ile Phe Gly Gln Ser Glu Ser Arg 1 5 10 44511PRTYersinia pestis 445Tyr Tyr Ser Val Thr Ala Gly Pro Val Phe Arg 1 5 10 44611PRTYersinia pestis 446Arg Gly Phe Glu Glu Ser Val Asp Gly Phe Lys 1 5 10 44712PRTYersinia pestis 447Val Gly Thr Trp Met Leu Gly Ala Gly Tyr Arg Phe 1 5 10

44817PRTYersinia pestis 448Ile Asn Glu Tyr Val Ser Leu Tyr Gly Leu Leu Gly Ala Gly His Gly 1 5 10 15 Lys 44917PRTYersinia pestis 449Tyr Glu Phe Asn Asn Asp Trp Gly Val Ile Gly Ser Phe Ala Gln Thr 1 5 10 15 Arg 45027PRTYersinia pestis 450Thr Ser Leu Ala Tyr Gly Ala Gly Leu Gln Phe Asn Pro His Pro Asn 1 5 10 15 Phe Val Ile Asp Ala Ser Tyr Glu Tyr Ser Lys 20 25 45117PRTYersinia pestis 451Ile Arg Glu Ala Ala Ala Ser Ile Ser Val Ile Ser Gln Asn Glu Leu 1 5 10 15 Arg

Claims

What is claimed is:

1. A method for treating an infection in a subject comprising: administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Yersinia spp., wherein the composition comprises: isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel, wherein the polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa are expressed by a Yersinia pestis at a greater level when incubated in media comprising an iron chelator than when grown in the media without the iron chelator; wherein the 94 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 961.44, 1167.49, 1257.64, 1371.63, 1383.64, 1408.71, 1520.82, 1668.86, 1685.79, 1713.78, 1716.81, 1796.92, 1832.92, 1844.91, 2218.12, and 2426.09 Da; wherein the 88 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 888.51, 926.46, 945.53, 960.54, 1171.60, 1176.57, 1289.64, 1332.67, 1357.66, 1403.74, 1418.68, 1507.73, 1578.78, 1672.80, 1735.83, 2400.17, and 2665.28 Da; wherein the 77 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 686.37, 784.49, 858.41, 952.50, 1140.65, 1155.66, 1170.64, 1197.57, 1402.71, 1408.68, 1482.73, 1522.71, 1550.77, 1617.74, 1674.78, 1745.84, 1787.92, 1819.96, 1851.87, 1940.75, 2013.02, 2017.97, 2056.96, 2168.01, 2169.10, 2426.25, 2457.00, and 2828.33 Da; wherein the 73 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 628.39, 748.43, 909.42, 930.51, 1291.71, 1370.81, 1440.70, 1478.71, 1586.83, 1604.86, 1640.87, 1654.77, 1705.82, 1707.83, 1757.91, 1796.97, 1856.01, 1912.94, 2004.03, 2072.02, 2155.08, 2301.07, 2395.11, 2484.12, 2557.36, 2557.36, 2675.42, 2983.33, 3161.51, 3184.52, 3424.79, and 3471.62 Da; and wherein the 64 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 713.42, 759.42, 773.40, 806.41, 919.48, 1023.50, 1051.53, 1102.55, 1164.56, 1186.57, 1199.60, 1281.67, 1394.68, 1444.73, 1479.70, 1545.80, 1667.72, 1692.82, 1730.85, 1789.81, 1904.85, 1968.90, 1981.02, 2009.89, 2027.02, 2058.99, 2162.17, 2363.13, 2377.30, 2819.49, and 2929.46 Da.

2. The method of claim 1 wherein the subject is a mammal.

3. The method of claim 2 wherein the mammal is a human.

4. The method of claim 1 wherein the Yersinia spp. is Y. enterocolitica or Y. pestis.

5. A method for treating a symptom in a subject comprising: administering an effective amount of a composition to a subject having an infection caused by a Yersinia spp., wherein the composition comprises: isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel, wherein the polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa are expressed by a Yersinia pestis at a greater level when incubated in media comprising an iron chelator than when grown in the media without the iron chelator; wherein the 94 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 961.44, 1167.49, 1257.64, 1371.63, 1383.64, 1408.71, 1520.82, 1668.86, 1685.79, 1713.78, 1716.81, 1796.92, 1832.92, 1844.91, 2218.12, and 2426.09 Da; wherein the 88 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 888.51, 926.46, 945.53, 960.54, 1171.60, 1176.57, 1289.64, 1332.67, 1357.66, 1403.74, 1418.68, 1507.73, 1578.78, 1672.80, 1735.83, 2400.17, and 2665.28 Da; wherein the 77 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 686.37, 784.49, 858.41, 952.50, 1140.65, 1155.66, 1170.64, 1197.57, 1402.71, 1408.68, 1482.73, 1522.71, 1550.77, 1617.74, 1674.78, 1745.84, 1787.92, 1819.96, 1851.87, 1940.75, 2013.02, 2017.97, 2056.96, 2168.01, 2169.10, 2426.25, 2457.00, and 2828.33 Da; wherein the 73 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 628.39, 748.43, 909.42, 930.51, 1291.71, 1370.81, 1440.70, 1478.71, 1586.83, 1604.86, 1640.87, 1654.77, 1705.82, 1707.83, 1757.91, 1796.97, 1856.01, 1912.94, 2004.03, 2072.02, 2155.08, 2301.07, 2395.11, 2484.12, 2557.36, 2557.36, 2675.42, 2983.33, 3161.51, 3184.52, 3424.79, and 3471.62 Da; and wherein the 64 kDa polypeptide has a mass fingerprint that includes polypeptide fragments having masses of 713.42, 759.42, 773.40, 806.41, 919.48, 1023.50, 1051.53, 1102.55, 1164.56, 1186.57, 1199.60, 1281.67, 1394.68, 1444.73, 1479.70, 1545.80, 1667.72, 1692.82, 1730.85, 1789.81, 1904.85, 1968.90, 1981.02, 2009.89, 2027.02, 2058.99, 2162.17, 2363.13, 2377.30, 2819.49, and 2929.46 Da.

6. The method of claim 5 wherein the subject is a mammal.

7. The method of claim 6 wherein the mammal is a human.

8. The method of claim 5 wherein the Yersinia spp. is Y. enterocolitica or Y. pestis.

9. The method of claim 5 wherein the symptom is diarrhea, enteritis, or a symptom of plague, or a combination thereof.